Bioactive peptides

ABSTRACT

The present invention provides a cytotoxic 7 to 25-mer peptide with three or more cationic residues which has one or more non-generic bulky and lipophilic amino acids, as well as esters, amides, salts and cyclic derivatives thereof as well as methods of preparing the peptides, pharmaceutical compositions containing them, and their use as medicaments, particularly as antibacterial or antitumor agents.

This application is a continuation of U.S. application Ser. No.09/798,026 filed Feb. 27, 2001 now U.S. Pat. No. 7,439,228, which acontinuation of International Application Serial No. PCT/GB99/02850filed Aug. 31, 1999, which claims priority of Great Britain ApplicationGB9818038.4 filed Aug. 28, 1998, the disclosures of which areincorporated herein by reference.

The present invention relates to bioactive peptides, more particularlyto peptides which have been modified to enhance their cytotoxicactivity.

A wide variety of organisms use peptides as part of their host defensemechanism, in vertebrates this supplements the highly specificcell-mediated immune system [Mor, a., Hani, K. and Nicolas, P. (1994) J.Biol. Chem. 269, 31635-31641. Boman, H. G. (1996) Scand. J. Immunol. 43,475-482]. Antimicrobial peptides have been isolated from species asdiverse as bacteria and mammals [Lehrer, R. I., Lichtenstein, A. K. andGanz, T. (1993) Ann. Rev. Immunol. 11, 105-128]. Generally, theseantibiotic peptides have a net positive charge and a propensity to formamphiphilic α-helix or β-sheet structures upon interaction with theouter phospholipid bilayer in bacterial cell membranes [Besalle, R.,Gorea, A., Shalit, J., Metger, J. W., Dass, C. Desiderio, D. M. andFridkin, M. (1993) J. Med. Chem. 36 1203-1209]. In most cases thedetailed molecular mechanisms of the antibiotic action are unknown,although some peptides categorised as class L (lytic) peptides arebelieved to interact with bacterial cell membranes, probably formingion-channels or pores [Ludtke, S. J., He, K., Heller, W. T., Harroun, T.A., Yang, L. and Huang, H. W. (1996) Biochemistry 35 13723-13728]leading to permeability changes and consequent cell lysis.

Magainins are antibacterial peptides from the skin of the frog Xenopuslaeris and are classified as class L antibiotics because theyspecifically lyse bacteria; other peptides such as mastroparans, a beevenom, lack this specificity as they lyse eukaryotic as well asprokaryotic cells and are called Class L Venoms [Tytler, E. M.,Anantharamaiah, G. M., Walker, D. E., Mishra, V. K., Palgunachari, M. N.and Segrest, J. P. (1995) Biochemistry 34 4393-4401]. Anti-bioticresistance exhibited by certain infectious microorganisms is anincreasing problems and there is always a need for new antibiotics.Anti-bacterial peptides such as the class L peptides are known and moreare being discovered, with the aim of finding a peptide which is highlycytotoxic and preferably specific for prokaryotic cells. There aredifferences in the structure and composition of lipid bi-layers betweeneukaryotes and prokaryotes and amongst prokaryotes themselves which meanthat different peptides will have widely differing specificities.

As well as magainins and mastroparans, host defense peptides have beenisolated from moths and flies (cecropins) and from Horseshoe crab. Thedirect action of these host defense peptides to repel predators, forexample as venoms, is clear. The search for peptides which exhibitantibiotic effects has lead to the identification of otherproteins/peptides which would not be expected to have cytotoxicproperties. One of these is lactoferrin, an iron transporter which alsoshows a weak antibacterial effect.

As well as searching for new antimicrobial peptides, more recently ithas been sought to enhance the activity of proteins or peptides withknown antimicrobial properties. This has been done in the case of bovinelactoferrin by digesting the native protein with gastric pepsin toproduce a peptide, lactoferricin B (LFB), which is much more active thanthe native bovine lactoferrin. LFB is a 25 residue peptide whichcorresponds to residues 17-41 of bovine lactoferrin. [Bellamy et al.(1992) Biochem. Biophys. Acta. 1121 pp 130 et seq.]. Structure-activitystudies have been carried out on magainins and it has been shown, forexample, that enhancement of helicity and of the cationic charge leadsto higher antibacterial activity [Chen, Y. H., Brown, J. H., Morell, J.L. and Huang, C. M. (1988) FEBS Letters 236, 462-466]. However, suchsequence modifications often result in higher hemolytic activity. It isthus an object of the present invention to prepare peptides and/orpeptide derivatives which have significant antibacterial activity butpreferably have low toxicity, i.e. little effect on normal eukaryoticcells, e.g. low hemolytic activity. While red blood cells may not betypical eukaryotic cells, they provide a convenient way of assaying fortoxicity and in any event are a type of cell which should not be lysedto a significant extent by therapeutic bioactive peptides.

It has been found that by increasing the bulk or lipophilic nature of apeptide, its bioactivity can be increased, in particular itscytotoxicity. Preferably, the bulk and lipophilicity of one or moreamino acid residues is increased.

Thus, according to the present invention is provided a cytotoxic 7 to 25mer peptide with three or more cationic residues which is optionallycapable of forming an amphiphatic α-helix and which either has one ormore non-genetic bulky and/or lipophilic amino acids or has at least a40% sequence homology with a known or natural cytotoxic peptide and oneor more extra bulky and/or lipophilic amino acids, as well as esters,amides, salts and cyclic derivatives thereof.

The % homology is preferably 50 or 60% or more, particularly 70 or 80%or more. For the purposes of the present invention, the term “sequencehomology” is not used to refer to sequence identity but to the presenceof either the same amino acid or one from the same functional group. Thestandard genetically coded amino acids can be grouped according to theircharacteristics, particularly of polarity and charge. Convenientgroupings are, glycine and alanine, serine, threonine, asparagine,glutamine and cysteine, lysine, arginine and histidine, aspartic acidand glutamic acid and valine, leucine, isoleucine, methionine,phenylalanine, tryptophan and tyrosine. Of the 20 standard (genetic)amino acids, valine, leucine, isoleucine, methionine, tyrosine,tryptophan and phenylalanine are intended to be covered by the term“bulky and/or lipophilic” amino acid, isoleucine, tryptophan andphenylalanine being preferred. Throughout this specification, the widelyused and understood three letter and one letter code for the 20 standardamino acids has been used. Replacement of an amino acid from one groupwith another amino acid in the same group is conveniently referred to asa “conservative substitution”. Such substitutions do not generallymaterially effect the properties of the peptides of the invention andwhere any peptide differs from another only by such substitutions, ifone peptide is a peptide according to the present invention thentypically the other peptide will also be a peptide according to theinvention.

According to a preferred aspect of the present invention is provided acytotoxic 7 to 25 mer peptide with three or more cationic residues whichis optionally capable of forming an amphipathic α-helix and which hasone or more non-genetic bulky and lipophilic amino acids, as well asesters, amides, salts and cyclic derivatives thereof.

Peptides incorporating a non-genetic bulky and lipophilic amino acidwill preferably exhibit an enhanced cytotoxic effect against bacterialor tumour cells while the toxicity of the peptides, e.g. their hemolyticactivity is reduced or only moderately increased as compared to thenative or original peptide.

It has surprisingly been found that amino acids or their derivatives ofa certain size can be used to provide modified peptides which areparticularly suitable for use as cytotoxic peptides. Thus, according tothe invention, by “non-genetic bulky and lipophilic amino acid” is meantany amino acid or amino acid derivative, which may be naturallyoccurring, but not one of the 20 standard genetically coded amino acids,whose R group (α-side chain) is preferably uncharged and has at least 7,preferably 8, more preferably 9 non-hydrogen atoms. Particularlypreferred non-genetic bulky and lipophilic amino acids will have atleast 12, preferably at least 18 non-hydrogen atoms in the R group. Byway of example, the R group of the amino acid phenylalanine has 7non-hydrogen atoms but as it is one of the genetically coded or‘standard’ amino acids, it does not fall within our definition of“non-genetic bulky and lipophilic amino acids”. The term ‘non-hydrogen’is used to indicate that hydrogen atoms are not included when countingthe number of atoms present in a group or molecule.

Preferably, the R group in the non-genetic bulky and lipophilic aminoacid will have at least 8 or 9 non-hydrogen e.g. carbon atoms, ideallyincluding a closed ring system, more preferably it should have at least2 closed rings of 5 or 6 atoms and conveniently these two rings arefused or bridged. The group may comprise only one ring which issubstituted by heavily branched alkyl groups which include more than onebranch site or one branch site which has 4 attachments to non-hydrogenatoms. The rings are formed of carbon atoms, optionally also includingnitrogen, oxygen or sulphur atoms. Particularly preferred amino acidscomprise a substituted or unsubstituted indole. The group shouldpreferably be three-dimensional. Preferred non-genetic bulky andlipophilic amino acids include adamantylalanine, 3-benzothienylalanine,4,4′-biphenylalanine, 3,3-diphenylalanine, homophenylalanine,2,6-dichlorobenzyltyrosine, cyclohexyltyrosine, 7-benzyloxytryptophan,tri-tert-butyltryptophan, homotryptophan, 3-(-anthracenyl)-L-alanine,L-p-iso-propylphenylalanine, L-thyroxine, 3,3′,5-triiodo-L-thyronine.

A lipophilic molecule is one which associates with its own kind in anaqueous solution, not necessarily because the interactions between thelipophilic molecules are stronger than between the lipophilic moleculeand water but because interactions between a lipophilic molecule andwater would destroy the much stronger interactions between the watermolecules themselves. It is therefore preferable that the R group of thenon-genetic bulky and lipophilic amino acid should not contain manypolar functional groups e.g. no more than 4, preferably 2 or less. Suchgroups would increase the binding interaction with the aqueoussurroundings and hence lower the lipophilicity of the molecule. Highlylipophilic groups thus being preferred. For example, a phenyl group as acomponent of a bulky and lipophilic group would be preferred to apyridyl group, even though they have the same number of non-hydrogenatoms and are of a similar overall size.

Suitable bulky and lipophilic amino acid residues will therefore includenaturally occurring and non-naturally occurring amino acids which havean R group as previously defined, e.g. adamantylalanine or any aminoacid, including genetically coded amino acids, whose R groups have beenmodified to provide a non-genetic bulky and lipophilic amino acid aspreviously defined.

Non-genetic bulky and lipophilic amino acids in this second categoryinclude modified tryptophan and phenylalanine residues, in particulartryptophan residues which have been substituted at the 1-, 2-, 5- and/or7-position of the indole ring, positions 1- or 2-being preferred. Avariety of other amino acid derivatives having a bulky and lipophiliccharacter are known to the skilled man and are intended to be includedwithin the term “non-genetic bulky and lipophilic amino acid”.

Suitable amino acids include thyroxine and the following commerciallyavailable amino acids and their derivatives:

L-3-benzothienylalanine, CAS=72120-71-9 (Synthetech),D-3-benzothienylalanine, CAS=111139-55-0 (Synthetech),L-4,4′-biphenylalanine (Synthetech), D-4,4′-biphenylalanine(Synthetech), L-4-bromophenylalanine, CAS=24250-84-8 (Synthetech),D-4-bromophenylalanine, CAS=62561-74-4 (Synthetech),L-2-chlorophenylalanine, CAS=103616-89-3 (Synthetech),D-2-chlorophenylalanine, CAS=80126-50-7 (Synthetech),L-3-chlorophenylalanine, CAS=80126-51-8 (Synthetech),D-3-chlorophenylalanine, CAS=80126-52-9 (Synthetech),L-4-chlorophenylalanie, CAS=14173-39-8 (Synthetech),D-4-chlorophenylalanine, CAS=14091-08-8 (Synthetech),D-3-cyanophenylalanine, CAS=57213-48-6 (Synthetech),D-3-cyanophenylalanine (Synthetech), L-4-cyanophenylalanine(Synthetech), D-4-cyanophenylalanine (Synthetech),L-3,4-dichlorophenylalanine, CAS=52794-99-7 (Synthetech),D-3,4-dichlorophenylalanine, CAS=52794-98-6 (Synthetech),L-3,3-diphenylalanine (Synthetech), D-3,3-diphenylalanine (Synthetech),L-homophenylalanine, CAS=943-73-7 (Synthetech), D-homophenylalanine,CAS=82795-51-5 (Synthetech), L-2-indanylglycine (Synthetech),D-2-indanylglycine (Synthetech), L-4-iodophenylalanine, CAS=24250-85-9(Synthetech), D-4-iodophenylalanine, CAS=62561-75-5 (Synthetech),L-1-naphthylalanine, CAS=−55516-54-6 (Synthetech), D-1-naphthylalanine,CAS=78306-92-0 (Synthetech), L-2-Naphthylalanine, CAS=58438-03-2(Synthetech), D-2-naphthylalanine, CAS=76985-09-6 (Synthetech),L-3-trifluoromethylphenylalanine, CAS=14464-68-7 (Synthetech),D-3-trifluoromethylphenyl-alanine (Synthetech),L-4-trifluoromethylphenylalanine, CAS=114926-38-4 (Synthetech),D-4-trifluoromethyl-phenylalanine, CAS=114872-99-0 (Synthetech),Boc-D-homophenylalanine (Neosystem Laboratoire), Boc-L-homophenylalanine(Neosystem Laboratoire), Fmoc-4-methyl-D-phenylalanine (NeosystemLaboratoire), Fmoc-4-methyl-L-phenylalanine (Neosystem Laboratoire),2,6-dichlorobenzyltyrosine, CAS=40298-71-3 (Senn Chemicals),Benzyltyrosine Fmoc (Senn Chemicals), Cyclohexyltyrosine Fmoc (SennChemicals), L-3,5-diiodotyrosine, CAS=300-39-0 (Senn Chemicals),D-3,5-diiodotyrosine (Senn Chemicals), L-3,5-dibromotyrosine (SennChemicals), D-3,5-dibromotyrosine (Senn Chemicals), L-t-butyltyrosine(Senn Chemicals), L-t-butyltyrosine (Senn Chemicals),N-Acetylhomotryptophan (Toronto Research), 7-Benzyloxytryptophan(Toronto Research), Homotryptophan (Toronto Research),3-(-Anthracenyl)-L-alanine Boc (or Fmoc) (Peninsula Laboratories),3-(3,5-Dibromo-4-chlorophenyl)-L-alanine (Peninsula Laboratories),3-(3,5-Dibromo-4-chlorophenyl)-D-alanine (Peninsula Laboratories),3-(2-Quinoyl)-L-alanine Boc (or Fmoc) (Peninsula Laboratories),3-(2-Quinoyl)-D-alanine Boc (or Fmoc) (Peninsula Laboratories),2-Indanyl-L-glycine Boc (Peninsula Laboratories), 2-Indanyl-D-glycineBoc (Peninsula Laboratories), L-p-t-butoxyphenylglycine Fmoc (RSP),L-2-t-butoxyphenylalanine Fmoc (RSP), L-3-t-butoxyphenylalanine Fmoc(RSP), L-homotyrosine, O-t-butyl ether Fmoc (RSP),L-p-t-butoxymethylphenylalanine Fmoc (RSP), L-p-methylphenylalanine Fmoc(RSP), L-p-ethylphenylalanine Fmoc (RSP), L-p-iso-propylphenylalanineFmoc (RSP), L-p-methoxyphenylalanine Fmoc (RSP),L-p(tBu-thio)phenylalanine Fmoc (RSP), L-p-(Trt-thiomethyl)phenylalanineFmoc (RSP), L-p-hydroxymethyl-phenylalanine, O-t-butyl (RSP),L-p-benzoylphenylalanine (Advanced ChemTech), D-p-benzoyl-phenylalanine(Advanced ChemTech), O-benzyl-L-homoserine Boc (Advanced ChemTech),O-benzyl-D-homoserine Boc (Advanced ChemTech), L-β-1-Naphthyl-alanine(Advanced ChemTech), D-β-1-Naphthyl-alanine (Advanced ChemTech),L-penta-fluorophenylalanine Boc (Advanced ChemTech),D-penta-fluorophenylalanine Boc (Advanced ChemTech),D-penta-fluorophenylalanine Fmoc (Advanced ChemTech),3,5-Diiodo-L-tyrosine Fmoc (Boc) (Advanced ChemTech), L-Thyroxine Na,CAS 6106-07-6 (Novabiochem), 3,3′,5-Triiodo-L-thyronine Na, CAS=55-06-1(Novabiochem).

Surprisingly, it has been found that standard chemical protecting groupswhen attached to an R group and thus increasing the bulk andlipophilicity of the residue can increase the bioactivity of peptides.Such protecting groups are well known in the art. Suitable protectinggroups which can significantly enhance anti-bacterial activity includePmc (2,2,5,7,8-pentamethylchroman-6-sulphonyl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulfonyl) and Pbf(2,2,4,6,7-pentamethyldihydrobenzofuransulfonyl), which may convenientlyincrease the bulk and lipophilicity of aromatic amino acids, e.g. Phe,Trp and Tyr. Also, the tert-butyl group is a common protecting group fora wide range of amino acids and is capable of providing non-geneticbulky and lipophilic amino acids as described herein, particularly whenmodifying aromatic residues. The Z-group (carboxybenzyl) is a furtherprotecting group which can be used to increase the bulk andlipophilicity of an amino acid to provide a peptide in accordance withthe invention.

Although the initial observation of increased bioactivity was as aresult of a serendipitous transfer of the protecting group Pmc withinthe peptide from the guanidino group of arginine to tryptophan, aminoacids such as Trp which carry the protecting group can be synthesiseddirectly and incorporated into the peptide.

This observation of the transfer of Pmc from Arg to Trp has beenobserved by Stierandova et al. in Int. J. of Peptide Science (1994) 43,31-38. Peptides in accordance with the invention can be made byutilising this transfer of the protecting group from Arg to Trp. Whenthese two amino acids are separated by 1-3 amino acids the transfer ofPmc is most efficient. Peptides according to the invention may thusconveniently comprise an amino acid carrying a protecting group, e.g.Trp with Pmc attached in the 2 position of the indole ring. The Pmcgroup may be attached to a Trp which has been added or to a Trp residuepresent in the original peptide. In a preferred embodiment of theinvention, peptides will incorporate one or more additional tryptophanresidues which can then be modified to further increase its bulky andlipophilic character and thus provide a peptide according to theinvention.

In the context of the present invention, “cyclic derivatives” refers topeptides which are cyclic as a result of one or more di-sulphidebridges. For some peptides incorporating two or cysteine residues, thiswill be the naturally occurring form and production of a linear peptidewill require the modification of the cysteine residues.

The non-genetic bulky and lipophilic amino acid may be present inaddition to the amino acids of the original sequence, which may itselfbe a naturally occurring peptide or fragment thereof or incorporateother modifications to a naturally occurring peptide or fragment or beentirely synthetic. Alternatively and preferably, the non-genetic bulkyand lipophilic amino acid may be in place of one of the amino acids inthe original sequence. When the amino acid is ‘added’, then all originalamino acids in the peptide remain. When the extra amino acid is“substituted”, it replaces one of the original amino acids, although areplacement may include modification of the existing residue to providea non-genetic bulky and lipophilic amino acid as previously defined.

The non-genetic bulky and lipophilic amino acid is preferably present inplace of another, naturally-occurring, non-essential amino acid. By“non-essential” is meant an amino acid whose presence is not requiredfor the peptide as a whole to demonstrate cytotoxic activity. Typically,the peptide prior to incorporation of a non-genetic bulky and lipophilicamino acid will exhibit some cytotoxic activity, this activity beingenhanced by the incorporation of a non-genetic bulky and lipophilicamino acid.

In a preferred embodiment of the invention, the non-genetic bulky andlipophilic amino acid will be present adjacent to or preferably in placeof a genetic bulky and lipophilic amino acid present in the originalpeptide. In other words, an already bulky and lipophilic amino acid ismade more bulky and lipophilic. This can be achieved by modification ofthe R group of the original amino acid or by replacing that amino acidwith a non-genetic amino acid. The genetically coded amino acids whichcan be considered bulky and/or lipophilic are defined previously. Thus,in a preferred embodiment of the present invention, the peptides willincorporate a non-genetic bulky and lipophilic amino acid in the form ofe.g. a modified tryptophan residue (e.g. Trp-Pmc) or e.g.tributyltryptophan residue in place of e.g. tryptophan or phenylalanine.

Preferably, the peptides of the invention will incorporate between 1 and5, e.g. 2 or 3 non-genetic bulky and lipophilic amino acids as hereindefined.

For any given cytotoxic peptide, suitable positions for incorporation ofnon-genetic bulky and lipophilic amino acids in order to increasecytotoxicity can be identified in a number of ways. As discussed above,“incorporation” may include modification of an existing residue. Analanine scan (involving sequential substitution of the amino acids withalanine) can be used to identify non-essential amino acids which couldbe substituted by a bulky and lipophilic amino acid or modified toincrease its bulk and lipophilicity. Alternatively, a candidate peptidewhich forms an amphiphatic α-helix can be represented as a ‘helicalwheel’ of residues and the cationic residues identified. These cationicresidues will form positively charged domains or regions within thethree-dimensional helical peptide structure and suitable positions forincorporation of or modification to provide non-genetic bulky andlipophilic amino acids are generally adjacent to or between suchcationic domains when viewed along the axis of the helical wheel.

It has even been found that peptides having enhanced antibacterialand/or antitumoural activity and preferably reduced toxicity can beprepared by moving a bulky and lipophilic amino acid from its positionin the original/native sequence to a region adjacent to the cationicsector, thus the overall amino acid composition of the peptide remainsunchanged. Such 7-25 mer peptides which have 3 or more cationic residuesand are capable of forming an amphipathic α-helix and which have anextra bulky and lipophilic amino acid adjacent to the cationic sector,said extra bulky and lipophilic amino acid being taken from another,non-preferred, position in the sequence constitute a further aspect ofthe present invention. In place of the bulky and lipophilic amino acidcan be put the residue from adjacent to the cationic sector which thebulky and lipophilic amino acid replaces or any other less bulky andlipophilic amino acid. Suitable bulky and lipophilic amino acids innon-preferred positions which can be moved into the region adjacent tothe cationic sector (preferred position) can be identified by e.g. analanine scan which identifies non-essential amino acids or by studying ahelical wheel arrangement, non-preferred positions typically beingopposite a cationic domain.

It has also been found that peptides having reduced toxicity but stillhaving reasonable antibacterial or anti-tumoural activity (i.e. havingenhanced selectivity) may be prepared by replacing a non-essentialhighly bulky and lipophilic amino acid such as tryptophan orphenylalanine with a less bulky and lipophilic amino acid e.g.isoleucine or leucine or even alanine or lysine. Generally, a“non-essential” bulky and lipophilic amino acid will be positioned onthe opposite side of the helix from the cationic sector, suchnon-essential bulky and lipophilic amino acids can be identified using ahelical wheel diagram or by an alanine scan. These peptides shouldnevertheless retain at least 3 bulky and lipophilic amino acids asherein defined. Thus, modified cytotoxic peptides having 7 to 25 aminoacids, at least three cationic residues and at least three bulky andlipophilic amino acids and being capable of forming an amphipathicα-helix, wherein one non-essential tryptophan or phenylalanine residuein the original/native sequence is replaced by a less bulky andlipophilic residue e.g. isoleucine or alanine constitute a furtheraspect of the present invention. Indolicin is a naturally occurringtryptophan rich peptide which may conveniently be modified in this wayto reduce its toxicity.

Other suitable sites for incorporation of a bulky and lipophilic aminoacid are positions at or near, preferably adjacent, to an existinglipophilic amino acid. Proximity is judged in terms of the secondaryrather than primary structure of the peptide. The techniques involved inperforming an alanine scan and in constructing helical wheel diagramsare well known in the art.

In the case of LFB(17-31) (a 15 amino acid fragment of LFB which lacksthe ten C-terminal residues), non-essential amino acids determined usingan alanine scan were Cys(3), Gln(7) and Gly(14), here the numbering isin absolute terms relating to the peptide itself. Analogs of LFB(17-31)wherein these amino acids are replaced by non-genetic bulky andlipophilic amino acids may be particularly effective. For modificationsto magainin peptides such as magainin 2, incorporation of non-geneticbulky and lipophilic amino acids at positions Phe(16) and Glu(19) may beparticularly effective.

In addition to the presence of one or more non-genetic bulky andlipophilic amino acid, the peptides according to the invention mayadvantageously incorporate further modifications. In particular,increasing the overall positive change of the peptide, for example byreplacing one or more naturally occurring amino acids, particularlynon-essential amino acids, with one or more positively charged residuessuch as lysine or arginine may further enhance the activity of thepeptide. “Positively charged” refers to the side chain (R group) of theamino acid residue which has a net positive charge at pH 7.0. In thecase of peptides for use as anti-tumour agents, where the peptide mayadvantageously be capable of forming α-helix, substitutions within thepeptide sequence which serve to lower the angle subtended by thecationic sector, i.e. the angle of the positively charged face of thehelix may further enhance activity. In fact, lowering the anglesubtended may have a greater impact on activity than the net positivecharge per se. Other residues may advantageously be replaced by alanine.Additional ‘genetic’ bulky and/or lipophilic amino acids as definedherein, e.g. Trp or Phe may also advantageously be incorporated.

Suitable peptides which can be modified to provide peptides inaccordance with the invention include all peptides such as themagainins, PGLa analogues, cecropins, defensins, melittin andlactoferrin, and class (L) lytic peptides generally etc. which are knownin their unmodified form to exhibit cytotoxic, particularlyanti-microbial activity. Further suitable peptides include those whichare not naturally occurring but have been synthesised and exhibitcytotoxic activity, such peptides include the modelines. In thiscontext, “unmodified” includes fragments obtained by digestion ofnaturally occurring proteins or peptides. New anti-bacterial proteinsand peptides are still being discovered and it is believed that thetechniques of the present invention have general applicability and couldbe applied simply, and with a reasonable chance of success, to peptideswhich are as yet unidentified but are subsequently characterised ascytotoxic, particularly as antimicrobial.

Particularly preferred peptides according to the present invention arethose which are based on fragments of lactoferrin, particularly thosebased on bovine lactoferrin (LFB) or fragments (e.g. LFB 17-31) thereofor the equivalent fragment of lactoferrin from other animals.

A particular advantage of the peptides of the present invention is theirsmall size, peptides having 15 or fewer amino acids being preferred,conveniently of 9 or 10 amino acids or less. One such effective smallpeptide is LFB(17-27) wherein the Lys28, Leu29, Gly30 and Ala31 from theC-terminal end of LFB(17-31) have been omitted. The peptides may beproduced by any known method, conveniently by enzymatic digestion orchemical cleavage of native peptides and subsequent modification or bydirect synthesis from the amino acid building blocks. The shorter thedesired peptide the better as far as manufacture is concerned,particularly for direct synthesis which is the preferred method ofmanufacture, as this limits the problems associated with chirality ofthe amino acids. In addition, short peptides are good for biodelivery.There is a growing demand for anti-biotics which can be administeredwithout the need for an injection, such as by inhalation and absorptionacross the blood capillaries of the nasal passages. A 10 mer peptidecould easily be administered in this way but peptides in excess of 25amino acids in length could not be delivered by inhalation.

It would also be desirable to increase the circulating half-life of thepeptide and this could be achieved by further modifying the peptides ofthe invention to include artificial amino acids as they are resistant toenzymatic breakdown. Long peptides are susceptible to breakdown byendopeptidases which cleave internally of the peptide, shorter peptideswould be less vulnerable to cleavage by endopeptidases and breakdown byexopeptidases, which attack the ends of a peptide, could be reduced byacetylating the N terminus and otherwise blocking the C terminus.

It has also been observed that the incorporation of enantio amino acidscan significantly increase the bioactivity of the peptides of theinvention and such peptides constitute a further preferred embodiment ofthe present invention. Excellent antimicrobial activity has been shownfor Enantio peptides which are the exact mirror image of the nativepeptide and Retro-Enantio peptides which adopt the same α-helicalconfirmation as the native peptide except the amide bonds point inopposite directions. Preferably, in accordance with the invention, suchpeptides will also incorporate a non-genetic bulky and lipophilic aminoacid as previously defined.

Enantio amino acids are also resistant to enzymatic breakdown and theresultant increase in half-life of the peptides may go some way toexplaining the enhanced anti-bacterial activity. Enantio amino acids areexpensive and this is a further reason why the relatively short peptidesof the present invention are particularly advantageous.

Further preferred peptides according to the invention thereforeincorporate a non-genetic bulky and lipophilic amino acid as previouslydefined and also comprise one or more D-amino acids, e.g. ⅓ or ½ or ⅔ ofthe amino acids are in the D form and these may be arranged in any waythroughout the sequence e.g. alternately with L amino acids.

By the term “capable” of forming an amphipathic α-helix is meant thatthe peptide may, in certain circumstances, form an α-helix. Peptides maynot necessarily have the α-helix as their natural configuration inaqueous media but are able, for example in the presence of helixproviding substances such as sodium dodecylsulphate (SDS),2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoroisopropanol (HFIP)or micelles other than SDS and cell membranes (artificial and natural)to form an α-helix or substantially α-helical structure. Circulardichroism may conveniently be used to test for the presence of anα-helix.

Of more importance than the formation of an α-helix is the fact that thepeptides are amphipathic, i.e. that the 2° structure of the peptide,whether it is α-helical or not, is amphipathic. This is evidenced by thegood activity of enantio peptides which do not form an α-helix in anyenvironment and peptides incorporating one or more D-amino acids, therequirement for an amphipathic α-helical conformation is thus not anessential requirement of the present invention.

In addition, the present invention relates to non-peptide compoundsshowing the same cytotoxic activity as their proteinaceous counterparts.Such petidomimetics or “small molecules” capable of mimicking theactivity of a protein or peptide are likely to be better suited for e.g.oral delivery due to their increased chemical stability. Such compoundswill include a part which corresponds to the “non-genetic bulky andlipophilic amino acid” as previously defined. More particularly, theywill include a group which corresponds to the R group of saidnon-genetic bulky and lipophilic amino acid, i.e. it has at least 7,preferably at least 9, non hydrogen atoms in the equivalent of the Rgroup, that group being uncharged and preferably comprising few polargroups.

It is now commonplace in the art to replace peptide or protein-basedactive agents e.g. therapeutic peptides with such peptidomimetics havingfunctionally-equivalent activity. Various molecular libraries andcombinatorial chemistry techniques exist and are available to facilitatethe identification, selection and/or synthesis of such compounds usingstandard techniques (Kieber-Emons, T. et al. Current Opinion inBiotechnology 1997 8: 435-441). Such standard techniques may be used toobtain the peptidomimetic compounds according to the present invention,namely peptidomimetic organic compounds which show substantially similaror the same cytotoxic activity as the peptides of the invention, e.g. asdescribed herein in the Examples.

A further aspect of the invention thus provides a biomimetic organiccompound based on the peptides of the invention, characterised in thatsaid compound exhibits cytotoxic, e.g. antibacterial or antitumouralactivity, at least the level exhibited by the peptides of the inventionas hereinbefore defined.

The term “cytotoxic” is intended to refer not only to an activityagainst prokaryotic cells but also against eukaryotic cells. Although incertain circumstances it is desirous to have a peptide which has a goodanti-bacterial activity but does not lyse or otherwise destroy the cellsof the patient, peptides within the scope of the present invention havebeen shown to have an anti-tumoural activity. The anti-tumoural activityof these peptides and medicaments containing them constitute furtheraspects of the present invention. Anti-tumoural activity includes thedestruction or reduction in size or number of benign or malignanttumours and the prevention or reduction of metastasis.

In general, lactoferrin derived peptides according to the inventionwhich have no non-genetic amino acids and have a good activity againsttumour cells will have 25-10, preferably 12-20 e.g. 18 amino acids.Peptides according to a non genetic bulky and lipophilic group andhaving good anti-tumoural activity will generally be shorter, with 7-20,preferably 10-20, more preferably 10-15 amino acids. By way of example,LFB 17-27 A7, M3, R2, 11W4,10, Y1-NH₂ PMC and LFB 18-24 R1,7 W2,3,6-NH₂PMC require only 50 and 38 μg/ml respectively to kill 50% of Meth Acells.

In general, peptides having good activity against tumours will be longerthan those exhibiting good anti-bacterial activity. Anti-bacterialpeptides will typically have 7 to 20, preferably 7 to 14, e.g. 8 or 9amino acids.

The anti-tumoural activity of the modified peptides is much better thancould be predicted merely from the fact that the peptides appear to havea lytic effect on bacterial cells. The observed lytic effect on tumourcells in vitro is powerful and tumour regression in mice is very rapid,occurring within 3-6 days. It appears that there is induction of animmunological memory, as inoculation of tumour cells in mice after thetreatment and regression of the original tumour did not give rise to anysecondary tumour growth.

Importantly, we have demonstrated regression of established tumours,even with unmodified LFB. In this context, “unmodified” refers also tofragments of LFB which exhibit this antitumoural activity, e.g.LFB(17-31). The peptide may be cyclic or linear, preferably cyclic. Theability to treat solid tumours is particularly useful when a tumour isunresectable. A further advantage is that the observed cytolytic effectin tumours is not species specific and thus the peptides have utility intreating human tumours.

Suitable doses for treatment of tumours with bioactive peptides will beknown to the skilled man and doses used in the animal experimentsdescribed herein can be used to estimate an appropriate dose for otheranimal and human patients. Administration of a peptide may be daily,more usually on alternate days or on every 3rd or 4th day. 1 to 10,typically 2 to 5 administrations may result in successful treatment.Similar treatment protocols will be used for treatment of bacterial orviral infections.

Peptides according to the invention will preferably be at least ascytotoxic as LFB (17-31. Some peptides according to the invention willbe more active in some respects (e.g. antitumoural) than LFB (17-31) butless active in other respects e.g. against E. coli. Some peptides may beless active but other properties e.g. a low hemolytic activity willrender them useful in certain applications.

The antibacterial activity of the peptides of the invention may manifestitself in a number of different ways. Certain modifications may resultin peptides which are bacteriostatic and others in peptides which arebacteriocidal. Advantageously, the majority of the peptides according tothe invention are bacteriocidal. Thus, inter alia, the invention alsoprovides a method of inhibiting the growth of bacteria comprisingcontacting the bacteria with an inhibiting effective amount of acytotoxic peptide according to the invention.

The term “contacting” refers to exposing the bacteria to a peptide sothat it can effectively inhibit, kill or lyse bacteria, bind endotoxin(LPS), or, permeabilize gram-negative bacterial outer membranes.Contacting may be in vitro, for example by adding the peptide to abacterial culture to test for susceptibility of the bacteria to thepeptide. Contacting may be in vivo, for example administering thepeptide to a subject with a bacterial disorder, such as septic shock.“Inhibiting” or “inhibiting effective amount” refers to the amount ofpeptide which is required to cause a bacteriastatic or bacteriacidaleffect. Examples of bacteria which may be inhibited include E. coli, Paeruginosa, E. cloacae, S. typhimurium and S. aureus. The method ofinhibiting the growth of bacteria may further include the addition ofantibiotics for combination or synergistic therapy. The appropriateantibiotic administered will typically depend on the susceptibility ofthe bacteria such as whether the bacteria is gram negative or grampositive, and will be easily discernable by one of skill in the art.

In addition, different modifications may enhance the antibacterialactivity against certain types of bacteria more than against othertypes. For example S. aureus is particularly susceptible to very largebulky and lipophilic groups, typically those having at least 12 or 18non-hydrogen atoms in the R group e.g. those peptides which incorporatea Pmc modified tryptophan residue. In addition, R groups which aresubstantially planar have good activity against E. coli while a more3-dimensional group of comparable lipophilicity is preferred forproducing good activity against S. aureus.

Although, as discussed above, the technique of enhancing activity byintroducing a non-genetic bulky and lipophilic amino acid is of generalapplicability to a wide variety of cytotoxic peptides, particularly toclass L (lytic) peptides, of particular interest are mammalian derivedpeptides, particularly peptides derived from lactoferrin, especiallylactoferricin. It has been found that the sequence of bovinelactoferricin (LFB 17-41) can be reduced by up to about 10 residues atthe C-terminal end, e.g. to LFB(17-31) without significant loss ofantibacterial activity. LFB 17-31=FKCRRWQWRMKKLGA. As well as bovinelactoferricins, we have identified the regions corresponding to LFB17-31 in man, LFH=TKCFQWQRNMRKVRG, goat, LFC=SKCYQWQRRMRKLGA, mice,LFM=EKCLRWQNEMRKVGG and pigs, LFP=SKCRQWQSKIRRTNP and such regions arealso suitable for manipulation according to the invention.

A variant of the effects of an increase in release, in which case theexcipients mentioned above for tablets may be used.

Organ specific carrier systems may also be used.

Injection solutions may, for example, be produced in the conventionalmanner, such as by the addition of preservation agents, such asp-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are thenfilled into injection vials or ampoules.

Nasal sprays which are a preferred method of administration may beformulated similarly in aqueous solution and packed into spraycontainers either with an aerosol propellant or provided with means formanual compression. Capsules containing one or several activeingredients may be produced, for example, by mixing the activeingredients with inert carriers, such as lactose or sorbitol, andfilling the mixture into gelatin capsules.

Suitable suppositories may, for example, be produced by mixing theactive ingredient or active ingredient combinations with theconventional carriers envisaged for this purpose, such as natural fatsor polyethyleneglycol or derivatives thereof.

Dosage units containing the compounds of this invention preferablycontain 0.1-10 mg, for example 1-5 mg of the peptides of the invention.The pharmaceutical compositions may additionally comprise further activeingredients, including other cytotoxic agents such as otherantimicrobial peptides. Other active ingredients may include differenttypes of antibiotics, cytokines e.g. IFN-γ, TNF, CSF and growth factors,immunomodulators, chemotherapeutics e.g. cisplatin or antibodies.

A yet further aspect of the present invention provides the therapeuticuse of the peptides of the invention as defined above i.e. the peptidesfor use as medicaments, e.g. antibacterions or antitumoural agents.Further aspects comprise a method of treating or lipophilicity ofcertain peptides discussed above has been observed and a further aspectof the present invention comprises a cytotoxic peptide of 15 amino acidsor less characterised in that it has an additional bulky/lipophilicgroup at one end. In respect of this aspect of the invention, thebulky/lipophilic group includes organic groups such as protectinggroups, especially Fmoc, Boc or other standard N terminal protectinggroups or branched, linear or cyclic alkyl groups of formulaCH₃(CH₂)_(n) wherein n is between 5 and 20, preferably between 8 and 14and most preferably 10 to 12 or branched, linear or cyclic acyl groupshaving between 6 and 21, preferably 9 and 15 and most preferably 11 to13 carbon atoms. For example, an LFB(17-31) peptide having aCH₃(CH₂)_(n) alkyl group at the N-terminal end had an up to 10 foldincrease in antibacterial activity. The groups are attached to N- orC-terminal or close, preferably adjacent, to N- or C-terminal residues.These groups may be attached to native amino acid residues, ornon-native amino acids carrying the bulky/lipophilic group may beincorporated into the peptide. The appropriate definition of “cytotoxicpeptide” is as discussed above.

The bulky/lipophilic nature of an amino acid and thus of a peptide canbe enhanced by N- or C-terminal modification and such modificationsresult in further peptides according to the present invention.

Thus peptides may, in addition to or instead of incorporating anon-genetic bulky and lipophilic amino acid as previously defined,therefore be modified at the N- and/or C-terminus.

More specifically, it has been found that peptides having antibacterialand/or antitumoral activity but a low toxicity can be made byincorporating N-terminal modifications which include a cyclic group,preferably a 5- or 6-membered ring which may be alkyl or aryl. Morepreferably the group which comprises the N-terminal modificationencompasses 2 or more fused rings one or more of which may be a5-membered ring e.g. adamantyl or Fmoc. It has surprisingly been foundthat groups which are three dimensional in character, such as thosewhich incorporate a fused ring system which does not lie in a singleplane have particularly advantageous properties.

Suitable molecules which could be used to modify the N-terminus include:

cis-Bicyclo[3.3.0]octan-2-carboxylic acid, [18209-43-3] (Aldrich);Abietic acid, [514-10-3] (Aldrich); Ursolic acid, [77-52-1] (Aldrich);(1,2-Methanofullerene C₆₀)-61-carboxylic acid, [155116-19-1] (Fluka);Dimethyl cubane-1,4-dicarboxylate, [29412-62-2] (Fluka);2-Norbornaneacetic acid, [1007-01-8] (Aldrich);4-Pentylbicyclo[2.2.2]octane-1-carboxylic acid, [73152-70-2] (Aldrich);3-Noradamantanecarboxylic acid, [16200-53-6] (Aldrich); 9-Fluoreneaceticacid, [6284-80-6] (Aldrich); cis-Decahydro-1-naphthol, [36159-47-4](Aldrich); 9-Ethyl-bicyclo[3.3.1]nonane-9-ol, [21915-33-3] (Aldrich);3-Quinuclidinol, [1619-34-7] (Aldrich); [[(1S)-endo]-(−)-Borneol,[464-45-9] (Aldrich); (1R,2R,3R,5S)-(−)-Isopinocampheol, [25465-65-0](Aldrich); Dehydroabietylamine [1446-61-3] (Aldrich);(±)-3-Aminoquinuclidine [6530-09-2] (Aldrich); (R)-(+)-Bornylamine,[32511-34-5]. (Aldrich); 1,3,3-Trimethyl-6-aza-bicyclo[3.2.1]octane[53460-46-1] (Aldrich); 1-Adamantylamine, [768-94-5] (Aldrich);9-Aminofluorene, [5978-75-6] (Aldrich); (1R)-(−)-10-Camphorsulfonicacid, [35963-20-3] (Aldrich); 5-Isoquinolinesulfonic acid, [27655-40-9](Aldrich); 2-Quinolinethiol, [2637-37-8] (Aldrich); 8-Mercaptomenthone,[38462-22-5] (Aldrich).

N-terminal modifications to provide peptides in accordance with theinvention will therefore typically comprise a bulky and lipophilic groupR which may be attached directly to the N-terminal amine to form amono-, di- and possibly cationic trialkylated N-terminal amine.Alternatively, the R group may be attached via a linking moiety e.g. acarbonyl group (RCO) e.g. adamantyl or benzyl, carbamate (ROCO) e.g.Fmoc, or a linker which forms urea (RNHCO) or (R₂NCO) or by a linkerwhich forms a sulfonamide, boronamide or phosphonamide. Sulfonamideforming linkers may be particularly useful when a more stable peptide isrequired. The bulky and lipophilic group R comprises a preferablysaturated cyclic group, more preferably a polycyclic group wherein thecyclic groups are fused or bridged.

Peptides incorporating such N-terminal modifications are particularlyeffective as anti-tumour peptides and surprisingly, the presence of acyclic, preferably multi-cyclic, N-terminal group provides peptides withan ability to kill tumour cells e.g. Meth A cells (from a fibrosarcoma)but have little cytotoxic activity against normal cells e.g. red bloodcells or normal fibroblast cells. This selectivity is, of course, highlydesirable in the in vivo treatment of established tumours. For example,cyclohexyl-LFB 17-31 at a concentration of 46 μg/ml killed 50% of Meth Acells (murine sarcoma cell line) but did not kill 50% of red blood cellsof fibroblasts even at a concentration of 1000 μg/ml.

Thus, according to a further aspect of the invention is provided acytotoxic 7 to 25 mer peptide with three or more cationic residues,which is optionally capable of forming an amphipathic α-helix and whoseN-terminus is modified by a cyclic group comprising 5 preferably 6 ormore non-hydrogen atoms, as well as esters, amides, salts and cyclicderivatives thereof. Pharmaceutical compositions containing suchmodified peptides together with a pharmaceutically acceptable diluent orcarrier and such peptides for use in methods of treatment, particularlyin the treatment or prevention of bacterial infections or as ananti-tumour agent (both in the destruction or reduction in size ornumber of benign or malignant tumours which may be ascites and in theprevention of metastasis) constitute further aspects of the presentinvention.

Particularly effective C-terminal modifications according to the presentinvention have also been investigated. Amidation of the C-terminus inorder to manipulate the overall charge of a peptide is known but it hasnow been found that larger C-terminal modifications, including theformation of esters, including thioesters or substituted primary andsecondary amides result in peptides with enhanced cytotoxic activity.The C-terminal modifying groups will advantageously contain more than 4,preferably 6, more preferably 8 or 10 or more non-hydrogen atoms andform e.g. a benzyl ester or amide. Other C-terminal groups includenaphthylamine, substituted aromatic amines such as phenyl-ethylamine,mono, di- or tri-amino alkyl groups etc., groups incorporating a cyclicgroup being preferred. Standard C-terminal protecting groups are alsosuitable as activity enhancing modifications.

C-terminal modifications to provide peptides in accordance with theinvention will therefore typically comprise a bulky and lipophilic groupR which may be attached directly to the C-terminal carboxy group to forma ketone. Alternatively, the R group may be attached via a linkingmoiety, e.g. (OR) which forms an ester at the C-terminus, (NH—R) or(NR₂, wherein the two R groups needs not be the same) which form primaryand secondary amide groups respectively at the C-terminus or groups(B—(OR)₂) which form boronic esters or phosphorous analogs. The bulkyand lipophilic group R preferably comprises at least 4 non-hydrogenatoms.

Thus, according to a further aspect of the invention is provided acytotoxic 7 to 25 mer peptide with three or more cationic residues,which is optionally capable of forming an amphipathic α-helix and whoseC-terminus is modified by an organic group comprising at least 4non-hydrogen atoms, as well as salts and cyclic derivatives thereof.Pharmaceutical compositions containing such modified peptides togetherwith a pharmaceutically acceptable diluent or carrier and such peptidesfor use in methods of treatment, particularly in the treatment orprevention of bacterial infections or as an anti-tumour agent (both inthe destruction or reduction in size or number of benign or malignanttumours which may be ascites and in the prevention of metastasis)constitute further aspects of the present invention.

Typically, the peptides of this aspect of the invention can berepresented by the following formula:

wherein X=a peptide of 7-25 amino acids in length incorporating 3cationic residues;

R═OR¹, SR¹ or R¹; and

R¹=alkyl, cycloalkyl, aminoalkyl or aryl optionally substituted byhydroxy, alkoxy, acyloxy, alkoxycarbonyloxy, amino, oxo or fluoro groupsand optionally interrupted by oxygen, nitrogen, sulphur or phosphorousatoms.

The substituted R¹ groups may be mono or polysubstituted. The term“acyl” as used herein includes both carboxylate and carbonate groups.

As used herein, the term “alkyl” includes a long or short chainstraight-chained or branched aliphatic saturated or unsaturatedhydrocarbon group. R¹ may contain up to 40 non-hydrogen atoms,preferably between 4 and 12, more preferably 6 to 10 such atoms.

Peptides according to the invention may comprise a non-genetic bulky andlipophilic amino acid as well as an N- and/or C-terminal modifying groupas defined herein. Peptides may include all three types of bulky andlipophilic groups but will preferably comprise two such groups.

A still further aspect of the present invention is a method of preparinga peptide having enhanced cytotoxic activity and/or improved selectivityfor target cell types which comprises incorporating a non-genetic bulkyand lipophilic amino acid into a 7 to 25 mer peptide with three or morecationic residues which is optionally capable of forming an amphiphaticα-helix.

Thus, the invention also provides a method of enhancing the cytotoxicityor selectivity of a 7 to 25 mer peptide with three or more cationicresidues by incorporating therein a non-genetic bulky and lipophilicamino acid.

A definition of non-genetic bulky and lipophilic amino acid is providedhereinbefore. As previously discussed “incorporating” may includemodification of an existing residue, or introduction of such a residueinto the peptide by addition or substitution, preferably substitution. Asynthetic method may be used whereby the non-genetic bulky andlipophilic amino acid is included in sequence in the growing peptide sono post peptide formation processing is required.

When, herein, we refer to a peptide having “enhanced” cytotoxicactivity, it is meant that the peptide which has been modified inaccordance with the invention has enhanced cytotoxicity against one ormore strains of bacteria or types of cancerous cells as compared to thepeptide without said modification. By “improved selectivity for targetcell types” is meant that the ratio of cytotoxic activity against targetcells as compared to non target cell types is increased. In other words,selectivity can be improved if, for example, the antibacterial activityof a peptide is the same before and after modification but the hemolyticactivity is decreased after modification. Similarly, useful peptidesaccording to the invention may be made even when hemolytic activityincreases, if the antibacterial or antitumoural activity increases by agreater amount. Selectivity may also refer to one type of bacteria overanother.

As described above, particularly active and useful peptides have beenprepared by incorporation of a non-genetic bulky and lipophilic aminoacid. It has also been found that increasing the bulk and lipophilicityof a peptide by incorporation of one or more additional “genetic” bulkyand lipophilic amino acids as previously defined may enhance activity.In particular, tryptophan rich analogs of peptides known to exhibit somecytotoxic activity have been shown to be effective as antimicrobialagents. Such analogs preferably have one or two tryptophan residuesreplacing other, non-essential residues.

Thus, in a further aspect of the present invention is provided acytotoxic 7 to 25 mer peptide, with three or more cationic residueswhich is optionally capable of forming an amphipathic α-helix and whichhas at least a 40% sequence homology with a known or natural cytotoxicpeptide and one or more extra genetic bulky and lipophilic amino acids(e.g. tryptophan), as well as esters, amides, salts and cyclicderivatives thereof.

The % homology is preferably 50 or 60% or more, particularly 70 or 80%or more. For the purposes of the present invention, the term “sequencehomology” is not used to refer to sequence identity but to the presenceof either the same amino acid or one from the same functional group.Suitable groupings for the standard amino acids are discussed above.

Sequence homology for such short peptides of the invention can mostsimply be calculated by comparing the two sequences, residue forresidue, to determine whether the two amino acids at positions 1, 2, 3etc. are the same or in the same group as previously defined. Thus, LFB(17-31)W3 has a 93.3% homology with LFB (17-31). Computer programs forcalculating sequence homology are also known in the art and these mayallow for additions (insertions) or deletions (gaps) in the sequence.Amino acid sequence homology may be determined using the BestFit programof the Genetics Computer Group (CGC) Version 10 Software package fromthe University of Wisconsin. The program uses the local homologyalgorithm of Smith and Waterman with the default values: Gap creationpenalty=8, Gap extension penalty=2, V Average match=2.912, Averagemismatch=−2.003. Such a program could therefore be used to assesshomology of the peptides of the invention with the native sequence,particularly if the modified peptide also incorporates gaps orinsertions. Such a program is most suited to establishing the alignmentbetween two sequences, again particularly when the modified sequenceincorporates gaps or insertions.

For such peptides which comprise only genetically coded amino acids,similarity of the modified peptides with a known or natural cytotoxicpeptide can be expressed by stringency of hybridisation of nucleic acidmolecules encoding the two sequences rather than % homology. In thiscase, the ssDNA molecule encoding the modified peptide should hybridisewith the ssDNA molecule complementary to the ssDNA molecule whichencodes the known or natural cytotoxic peptide. Nucleic acid moleculesencoding the peptides of the invention constitute further aspects of thepresent invention.

Sequences which “hybridise” are those sequences binding (hybridising)under non-stringent conditions (e.g. 6×SSC, 50% formamide at roomtemperature) and washed under conditions of low stringency (e.g. 2×SSC,room temperature, more preferably 2×SSC, 42° C.) or conditions of higherstringency (e.g. 2×SSC, 65° C.) (where SSC=0.15M NaCl, 0.015M sodiumcitrate, pH 7.2).

Preferably, the sequences will hybridise under conditions of higherstringency as defined above, or but for the degeneracy of the code, thesequences would hybridise under high stringency conditions.

Preferably, the peptides will include 1 or 2 additional genetic bulkyand lipophilic amino acids and may otherwise be identical to the knowncytotoxic peptide or incorporate only conservative substitutions.

In a further aspect, the invention provides a method of enhancing thecytotoxic activity of a peptide of 7 to 25 amino acids in length, whichhas three or more cationic residues and is optionally capable of formingan amphipathic α-helix which comprises introducing by addition orsubstitution, preferably substitution, a genetic bulky and lipophilicamino acid (e.g. tryptophan). By way of example, magainin derivedpeptides incorporating additional tryptophan residues and exhibitingenhanced activity are disclosed herein. Polynucleotides which encodethese peptides of the invention constitute further aspects of theinvention.

When the peptides of the invention incorporating a non-genetic bulky andlipophilic amino acid are derived from known or naturally occurringcytotoxic peptides or fragments thereof, they will preferably have thesame degree of homology with the known or naturally occurring peptide asis discussed above.

Peptides, particularly those wherein the bulky and lipophilic R group asdefined herein is a modified side chain of a ‘genetic’ amino acid, maybe expressed in prokaryotic and eukaryotic hosts by expression systemswell known to the man skilled in the art. Methods for the isolation andpurification of e.g. microbially expressed peptides are also well known.

If a bacterial host is chosen for expression of a peptide, it may benecessary to take steps to protect the host from the expressedanti-bacterial peptide. Such techniques are known in the art and includethe use of a bacterial strain which is resistant to the particularpeptide being expressed or the expression of a fusion peptide withsection at one or both ends which disable the antibiotic activity of thepeptide; the fusion peptide can then be cleaved. In any event, theactivity of the expressed peptide may be low, only enhanced to reallycytotoxic levels by the post-synthetic modification to provide a peptideaccording to the invention e.g. addition of Pmc.

The peptides of the invention may be directly synthesised in anyconvenient way. Generally the reactive groups present (for exampleamino, thiol and/or carboxyl) will be protected during overallsynthesis. The final step in the synthesis will thus be the deprotectionof a protected derivative of the invention. As discussed above, certainpeptides of the invention will carry a ‘protecting group’ as this isresponsible for enhanced cytotoxicity.

In building up the peptide, one can in principle start either at theC-terminal or the N-terminal although the C-terminal starting procedureis preferred. The non-genetic amino acid can be incorporated at thisstage as the sequence is extended or as a result of a post-syntheticmodification.

Methods of peptide synthesis are well known in the art but for thepresent invention it may be particularly convenient to carry out thesynthesis on a solid phase support, such supports being well known inthe art.

A wide choice of protecting groups for amino acids are known andsuitable amine protecting groups may include carbobenzoxy (alsodesignated Z) t-butoxycarbonyl (also designated Boc),4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr) and9-fluorenylmethoxy-carbonyl (also designated Fmoc). It will beappreciated that when the peptide is built up from the C-terminal end,an amine-protecting group will be present on the α-amino group of eachnew residue added and will need to be removed selectively prior to thenext coupling step.

Carboxyl protecting groups which may, for example be employed includereadily cleaved ester groups such as benzyl (Bzl), p-nitrobenzyl (ONb),pentachlorophenyl (OPClP), pentafluorophenyl (OPfp) or t-butyl (OtBu)groups as well as the coupling groups on solid supports, for examplemethyl groups linked to polystyrene.

Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt) andacetamidomethyl (Acm).

A wide range of procedures exists for removing amine- andcarboxyl-protecting groups. These must, however, be consistent with thesynthetic strategy employed. The side chain protecting groups must bestable to the conditions used to remove the temporary α-amino protectinggroup prior to the next coupling step.

Amine protecting groups such as Boc and carboxyl protecting groups suchas tBu may be removed simultaneously by acid treatment, for example withtrifluoroacetic acid. Thiol protecting groups such as Trt may be removedselectively using an oxidation agent such as iodine.

Peptides according to the invention may be prepared by incompletedeprotection to leave groups which enhance the cytotoxic activity of thepeptides. Alternatively, modified R and N- and C-terminal groups may beprepared after synthesis of the peptide and associated deprotection.

A particularly preferred method involves synthesis using amino acidderivatives of the following formula: Fmoc-amino acid-Opfp.

The present invention also provides pharmaceutical compositionscontaining the peptides of the invention as defined above together witha physiologically acceptable diluent, carrier or excipient. Suitablediluents, excipients and carriers are known to the skilled man. Thepeptides of the invention for use in methods of treatment particularlyin the treatment or prevention of bacterial infections or as ananti-tumour agent, both in the destruction or reduction in size ornumber of benign or malignant tumours which may be ascites and in theprevention of metastasis) constitute further aspects of the presentinvention.

The compositions according to the invention may be presented, forexample, in a form suitable for oral, nasal, parenteral, intravenal,intratumoral or rectal administration.

As used herein, the term “pharmaceutical” includes veterinaryapplications of the invention.

The compounds according to the invention may be presented in theconventional pharmacological forms of administration, such as tablets,coated tablets, nasal sprays, solutions, emulsions, liposomes, powders,capsules or sustained release forms. The peptides of the invention areparticularly suitable for topical administration, e.g. in the treatmentof diabetic ulcers. Conventional pharmaceutical excipients as well asthe usual methods of production may be employed for the preparation ofthese forms. Tablets may be produced, for example, by mixing the activeingredient or ingredients with known excipients, such as for examplewith diluents, such as calcium carbonate, calcium phosphate or lactose,disintegrants such as corn starch or alginic acid, binders such asstarch or gelatin, lubricants such as magnesium stearate or talcum,and/or agents for obtaining sustained release, such ascarboxypolymethylene, carboxymethyl cellulose, cellulose acetatephthalate, or polyvinylacetate.

The tablets may if desired consist of several layers. Coated tablets maybe produced by coating cores, obtained in a similar manner to thetablets, with agents commonly used for tablet coatings, for example,polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxideor sugar. In order to obtain sustained release or to avoidincompatibilities, the core may consist of several layers too. Thetablet-coat may also consist of several layers in order to obtainsustained preventing bacterial infections in a patient comprising theadministration to said patient of one or more of the peptides of theinvention and a method of treating tumours in a patient comprising theadministration of one or more of the peptides of the invention. Thetreatment of tumours includes the destruction or reduction in size ornumber of benign or malignant tumours which may be ascites and theprevention of metastasis.

A still further aspect of the present invention comprises the use of oneor more of the peptides of the invention in the manufacture of amedicament for treating bacterial infections or tumours.

Anti-bacterial agents such as the peptides of the present invention havea wide variety of applications other than as pharmaceuticals. They canbe used, for example, as sterilising agents for materials susceptible tomicrobial contamination. The peptides of the invention exhibit broadantimicrobial and antibiotic activity and thus are also suitable asanti-viral and anti-fungal agents which will have pharmaceutical andagricultural applications and as promoters of wound healing orspermicides. All of these uses constitute further aspects of theinvention.

The peptides, when used in topical compositions, are generally presentin an amount of at least 0.1%, by weight. In most cases, it is notnecessary to employ the peptide in an amount greater than 1.0%, byweight.

Anti-tumour peptides may be administered in combination, possibly insynergistic combination with other active agents or forms of therapy,for example administration of a peptide according to the invention maybe combined with chemotherapy, immunotherapy, surgery, radiation therapyor with the administration of other anti-tumour peptides.

In employing such compositions systemically (intra-muscular,intravenous, intraperitoneal), the active peptide is present in anamount to achieve a serum level of the peptide of at least about 5ug/ml. In general, the serum level of peptide need not exceed 500 ug/ml.A preferred serum level is about 100 ug/ml. Such serum levels may beachieved by incorporating the peptide in a composition to beadministered systemically at a dose of from 1 to about 10 mg/kg. Ingeneral, the peptide(s) need not be administered at a dose exceeding 100mg/kg.

Those peptides exemplified herein represent preferred peptides accordingto the invention. Any peptide whose specific sequence is disclosedherein, particularly those peptides which are more active againstbacterial cells than LFB 17-31, constitute a further aspect of thepresent invention.

Some of the preferred non-genetic bulky and lipophilic amino acidsincorporated into the peptides of the invention include substitutedtryptophans which provide an increase in bulk and lipophilicity and asignificant increase in bioactivity. Substitutions have been made at the1-position (or the indole N-position) and the adjacent 2-position andthese new compounds, described in Example 2 constitute a still furtheraspect of the present invention. New 1-substituted tryptophans include1-benzyl and 1-tosyl tryptophan.

The following novel, 2-substituted Tryptophan residues have been made,Z-Trp (2-nitrophenylsulfenylchloride)-OH and oxides thereof andZ-Trp(2-Pmc)-OH wherein Z is a protecting group, e.g. Fmoc. Method II ofExample 2E is a newly devised synthetic route suitable for thepreparation of a range of 2-sulfones and constitutes a further aspect ofthe present invention. Therefore, we further provide a method ofpreparing tryptophan residues substituted at the 2-position of theindole ring which comprises transferring the group with which thetryptophan will be substituted from a guanidyl containing group to anN-protected tryptophan. Preferably the guanidyl containing group is anarylalkyl or alkyl guanidyl group, most preferably it is aphenylethylguanidyl group. Preferably the N-protecting group is Fmoc andpreferably the tryptophan substituting group is Pmc.

LFB 17-41 whose cysteine residues have been blocked by pyridylethylationor acetamido-methylation but incorporate no further bulky and lipophilicamino acids are not per se peptides of the invention. Howeverpharmaceutical compositions comprising these peptides as well as use ofthe peptides as therapeutic agents as herein described constitutefurther aspects of the present invention.

The invention will now be described with reference to the followingnon-limiting examples in which.

FIG. 1 shows the amino acid sequence and charge at pH 7 for syntheticlactoferricins from different species;

FIG. 2 shows the effects of linear and cyclic lactoferricin B on a MethA fibrosarcoma cell line in vitro after 24 hours incubation;

FIG. 3 shows the effects of different LFB derivatives on Meth A cells invitro after ½ hour incubation, +=pmc-modified; −=unmodified;

FIG. 4 shows the effects of different LFB derivatives on Meth A cells invitro after 4 hours incubation, +=pmc-modified; −=unmodified;

FIG. 5 shows the effects of pmc modified retro LFB 17-31(+), Fmoc LFB17-31(A8) and LFB 17-31 on Meth A cells in vitro after ½ hour. RPMI wasused as negative control and Triton 100× as positive control.Concentrations are in mg/ml;

FIG. 6 shows the effects of pmc modified retro LFB 17-31(+), Fmoc LFB17-31(A8) and LFB 17-31 on Meth A cells in vitro after 4 hours. RPMI wasused as negative control and Triton 100× as positive control.Concentrations are in mg/ml;

FIG. 7 shows the dose response on human promyelotic leukemia cell lineHL 60 after 4 hours. HL 60 cells, 1×10⁴ were incubated with peptides 50,30, 20, 10, 5, 1 μg, 1000-20 μg/ml in 2 hours and coloured with MTT;

FIG. 8 shows inhibition of tumor growth; Meth A tumor cells (5×10⁷cells) were inoculated on day 1 and treated on day 7 and day 10 with 0.5mg (1 mg of P1) of the different peptides;

FIG. 9 shows the effect of D-LFB (17-31) A7 Pmc-NH₂ on B16F10 murinemelanoma.

FIG. 10 shows the size of tumours established in Bulb/c mice who arereinoculated with Meth A cells after successful treatment with cLFB. Themice were not treated with cLFB or other peptides in the study, thussome form of adaptive immunity is shown. Reinoculation of Meth A cells 1month after the LFB-treatment of Meth A tumours.

EXAMPLE 1 Human, Bovine, Murine and Caprine Lactoferrin Derived Peptides

A) MIC (Minimum Inhibitory Concentration) Tests

The bacterial strains used were: Escherichia coli ATCC 25922 andStaphylococcus aureus ATCC 25923. All strains were stored at −70° C. Thebacteria were grown in 2% Bacto Peptone water (Difco 1807-17-4). Alltests were performed with bacteria in mid-logarithmic growth phase.Determination of the minimum inhibitory concentration (MIC) of thepeptides for bacterial strains were performed in 1% Bacto Peptone water.A standard microdilution technique with an inoculum of 2×10⁶ CFU/ml wasused. All assays were performed in triplets. Since the peptides arepositively charged and therefore could adhere to the plastic wells, wecontrolled the actual concentration of the peptides in the solution byHPLC. There was no difference between the concentration of the peptidesbefore or after adding the solution to the plastic wells.

B) Synthesis of Peptides

Initially, the lactoferricin B used was a gift from Wayne Bellamy(Nutritional Science Laboratory, Morinaga Milk Industry Co. Ltd, Japan).Later in the study the peptides were synthesised with a 9050 PlusPepSynthesizer (Milligen). All peptides were synthesised on solid phaseby use of fluorenylmethoxycarbonyl (Fmoc) chemistry. Cysteines incystein containing peptides were protected with acetamidomethyl groupsto prevent disulfide bridge formation. The peptides were analysed andpurified by reversed phase HPLC on a Waters 600E chromatograph(Millipore) with UV detection at 254 nm. The fractions purified on HPLCwere analysed on a liquid chromatography-mass spectrometer (LC-MS) withelectrospray interface (Fisons VG Quattro) or/and with Fast AtomBombardment Mass Spectrometry (FAB-MS) (Fisons VG Tribrid).

Structure of the Lactoferricins

The structure of human lactoferrin is determined to 2.8 and 2.2 Åresolution by X-ray crystallography. Human lactoferricin (LFH) consistsof residues 1-47 of human lactoferricin. LFH contains two peptidefragments; one consisting of residues 12-47 cyclised with a disulfidebridge between Cys20 and Cys37, the second fragment (residues 1-11) isconnected to the 12-47 fragment through a disulfide bridge between Cys10and Cys46. In the human lactoferrin structure, the correspondingresidues comprises a β-strand (residues 4-11), an α-helix (residues12-29), a turn (residues 30 and 31), followed by a β-strand (residues31-47) [Day, C. L., Anderson, B. F., Tweedie, J. W. and Baker, E. N.(1993) J. Mol. Biol. 232, 1084-1100]. Bovine lactoferricin (LFB) withonly 25 residues (residues 17-41) in a single chain is structurally muchsimpler than LFH.

Antibiotic Activity of Synthetic Lactoferricins with Sequences fromDifferent Species

The amino acid sequence of lactoferrins from goat [Provost, F. L.,Nocart, M., Guerin, G. and Martin, P. (1994) Biochem. Biophys. Res.Commun. 203, 1324-1332] and mouse [Pentecost, B. T. and Teng, C. T.(1987) J. Biol. Chem. 262 10134-10139] have been determined and showhigh sequence homology with both the human and the bovine lactoferrins.The residues engaged in the helix-turn-strand motif can easily beidentified in the sequence as shown in FIG. 1. As LFB is moreantibacterial than LFH, the residues corresponding to LFB (17-41) werechosen in the amino acid sequence of human, murine and caprinelactoferrin to prepare analogous lactoferricin peptides; LFH (18-42),LFM (17-41) and LFC (17-41) respectively. The disulfide bridge is notessential for antibiotic activity in bovine and human lactoferricin[Bellamy et al. (1992)] and all peptides were prepared with ACMprotection of the cysteine residues to avoid cyclisation or oxidation.

The antibacterial activities of the synthetic lactoferricins expressedas MIC are compiled in Table 1 which shows that LFB (17-41) displayedthe most significant antibacterial activity against E. coli and S.Aureus.

TABLE 1 Minimum inhibitory concentration (MIC) in μg/ml (μM) ofsynthetic lactoferricins on E. coli ATCC 25922 and S. aureus ATCC 25923.E. coli S. aureus ATCC 25922 ATCC 25923 Peptide MIC MIC LFH(18-42) >200 >200 LFB (17-41) 30 30 LFM (17-41) >200 >200 LFC (17-31)750 1000 LFB (14-31) 70 (28) 200 (80) LFB (17-31) 40 (20) 100 (50) LFB(18-31) 80 (43) 200 (108) LFB (19-31) 200 (120) >250 (150) LFB (20-31)100 (62) 200 (124) LFB (17-31) K17 60 (30) 100 (50) LFB (17-31) F20 20(10) 200 (100) LFB (17-31) 20 (10) 200 (100) K17, F20 LFB analogs withdifferent chain length

A property considered to be important in determining the antibacterialactivity of linear peptides, is their ability to adopt helicalstructures. In the intact lactoferrin protein, residues 14-28 arelocated in an α-helix, residues 29-31 comprise a turn and residues 32-41are in a β-strand. We therefore anticipated that the antibacterialeffect of the lactoferricins could originate from the part of thesequence that participates in the helix of the intact protein. As thebovine lactoferricin sequence, LFB (17-41), was the only peptide withsignificant antibacterial property, we chose to prepare a shortervariety of the bovine peptide, LFB (17-31), containing both the helixand turn residues of the protein, while the 10 residues encompassing thestrand were removed. Despite the fact that LFB (17-31) has a lower netcharge (FIG. 1) than LFB (17-41) and LFC (17-41), it still retains mostof the antibacterial effect as shown in Table 1. These findings indicatethat even if the overall charge is important, it is not sufficient forantibacterial activity.

EXAMPLE 2 Preparation of Novel Substituted Tryptophans

In the following Examples and throughout the text the following generalformula: Z—XX(n-y)-OH refers to a substituted amino acid (XX) whereinthe NH₂ group of the amino acid is Z-protected, the amino acid isy-substituted at the n position and the COOH group of the amino acid isfree.

A) Preparation of Ac-Trp(1-Tos)-OH

Experimental:

A mixture of Ac-Trp-OEt (0.19 g, 0.69 mmol), tosyl chloride (0.20 g,1.04 mmol), tetrabutylammonium hydrogensulfate (2 mg, 0.01 equiv.) andNaOH (0.07 g, 1.73 mmol) in dichloromethane was stirred at roomtemperature for 2.5 hours. To the reaction mixture was added diluted HCluntil a pH of 2-3 was reached and then washed with water. To the organicphase was added a diluted base and the aqueous phase was extracted withdichloromethane, acidified and again extracted with dichloromethane.

¹H NMR (CDCl₃): δ 1.89 (s, 3H), 2.24 (s, 3H), 3.1-3.35 (m, 2H), 4.87 (m,1H), 6.63 (d, 1H), 7.1-7.3 (m, 4H), 7.46 (m, 2H), 7.68 (d, 2H), 7.89 (d,1H), 9.34 (s, broad, 1H).

MS (EI): m/z 382(10%), 284 (84%), 157 (8%), 155 (61%), 130 (26%), 129(24%).

Materials:

Ac-Trp-OEt Prepared according to procedure described under <<Peparationof diacetyltryptophan ethyl ester>>, Bodanszky, M and Bodanszky A, ThePractice of Peptide Synthesis (1994) p30; Vogel's Textbook of PracticalOrganic Chemistry 5th Ed. (1989) p.1273.B) Preparation of Fmoc-Trp(1-Benzyl)-OH

Boc-Trp(1-Benzyl)-OH¹:

Dimethyl sulfoxide (7 ml) was added to potassium hydroxide (0.73 g, 13mmol) (crushed pellets) and the mixture was stirred for 5 min.Boc-Trp-OH (1 g, 3.3 mmol) was then added and the mixture stirred for 1hour. Benzyl bromide (1.13 g, 6.6 mmol) was added and the mixture cooledbriefly and stirred for a further 20 hours before water (20 ml) wasadded. The mixture was extracted with diethyl ether (3×20 ml). The pH ofthe combined aqueous phases was adjusted to 2-3 by addition of 1M HCl(20 ml) and extracted with diethyl ether (3×20 ml). Each extract waswashed with water (3×20 ml). The combined diethyl ether phases weredried with MgSO₄ and the solvent removed under reduced pressure. Theproduct was isolated as white crystals (0.89 g, 2.3 mmol). Yield 69%.

¹H NMR (CDCl₃): δ 1.41 (s, 9H), 3.33 (m, 2H), 4.64 (m, 1H), 5.02 (m,1H), 5.24 (s, 2H), 6.95 (s, 1H), 7.01-7.38 (m, 8H), 7.59 (d, J=7.7 Hz,1H)

H-Trp(1-Benzyl)-OH:

Boc-Trp(1-Bn)-OH was dissolved in 98% TFA and stirred for 3 hours atroom temperature. Then the solvent was removed under reduced pressure.The product was isolated as an oil and used without furtherpurification.

Fmoc-Trp (1-Benzyl)-OH:

H-Trp(1-Bn)-OH (1.90 g, 6.5 mmol) was dissolved in a 10% solution ofNa₂CO₃ in water (21 ml, 20 mmol). Dioxane (15 ml) was added and themixture was stirred in an ice-water bath. 9-Fluorenylmethylchlorocarbonate (1.69 g, 6.5 mmol) was added in small portions andstirring was continued at ice-water bath temperature for 4 hours andthen at room temperature for 8 hours. The reaction mixture was pouredinto water (400 ml) and extracted with ether (3×200 ml). The combinedether phases were dried with MgSO₄ and the solvent removed under reducedpressure. The product was purified by chromatography on silica gel insolvent A (Solvent A=Ethylacetate Methanol=4:1). After purification theproduct was obtained as a white crystalline compound. The yield was 2.42g (72%).

¹H NMR (400 MHz, CDCl₃): δ 3.34 (m, 2H), 4.18 (m, 1H), 4.37 (m, 2H),4.78 (s, 1H), 5.19 (s, 2H), 5.31 (d, 1H), 6.91-7.74 (m, 19H).

Materials:

Boc-Trp-OH BACHEM no A-2360 Fmoc-ONSu Fluka no 46920 Trifluoroaceticacid Fluka no 91700/KEBO no 1.8341-100

-   Reference 1: Heaney, H., and Ley, S. V. J. Chem. Soc.    Perkin 1. (1973) 499-500    C) Preparation of Fmoc-Trp(2-Nps)-OH

To a solution of 2.0 g (4.7 mmole) Fmoc-L-tryptophan in 12 ml dioxane,0.87 g (4.6 mmole) of 2-nitrophenyl-sulfenylchloride (2-Nps-Cl) in 25 mldioxane was added under stirring at room temperature. After standing for3 days, 50 ml ethyl ether was added to the reaction mixture and thesolvent was evaporated. The product was purified by chromatography onsilica gel in solvent A (Solvent A=Chloroform:Ethanol:Neptane—1:1:1).R_(f) 0.43. After purification the product was obtained as ayellow-brown crystalline compound. The yield was 2.5 g (89%).

HPLC (C18): t_(R) 8.3 min, 85-100% B in 20 min. (A: H₂O+0.1% TFA; B:CH₃CN+0.1% TFA).

¹H NMR (DMSO-d₆): δ 3.16 (m, 1H), 3.38 (m, 1H), 4.00-4.10 (m, 3H), 4.19(m, 1H), 6.72 (d, J=8.1 Hz, 1H), 7.03 (t, J=7.3 Hz, 1H), 7.18 (t, 1H),7.22-7.49 (m, 7H), 7.60 (dd, J=7.3 and 12.1 Hz, 2H), 7.86 (m, 3H), 8.24(d, J=8.1 Hz, 1H), 11.51 (s, 1H).

After incorporation of Fmoc-Trp(2-Nps)-OH into a peptide, MSelectrospray analysis confirmed the expected molecular weight.

Materials:

Fmoc-Trp-OH BACHEM No B-1445/ SENN No 02019 2-Nitrophenylsulfenylchloride Fluka No 73740D) Oxidation of Fmoc-Trp(2-Nps)-OH

To a solution of 1.12 g (1.9 mmol) Fmoc-Trp(2-Nps)-OH in 15 ml glacialacetic acid, was added 12 ml 30% H₂O₂ under stirring at roomtemperature. The reaction mixture was heated for 2 hours at 65° C. Theprecipitate was collected, added water and lyophilised. The yield was0.59 g (52%). The product was obtained as a yellow crystalline compound.

HPLC (C18): t_(R) 6.4 min, 85-100% B in 20 min. (A: H₂O+0.1% TFA; B:CH₃CN+0.1% TFA).

¹H NMR (DMSO-d₆): δ 3.25 (dd, J=9.0 and 14.5 Hz, 0.5H), 3.54 (m, 1H),3.77 (dd, J=5.9 and 14.3 Hz, 0.5H), 4.01-4.26 (m, 3H), 4.32 (m, 0.5H),4.40 (m, 0.5H), 7.00-7.98 (m, 15H), 8.23 (m, 1H), 8.35 (m, 1H), 8.56 (d,J=8.1 Hz, 1H), 11.08 (s, 0.5H), 11.17 (s, 0.5H)

After incorporation of Fmoc-Trp(2-NpsO₂)—OH into a peptide, MSelectrospray analysis revealed that the oxidation of Fmoc-Trp(2-Nps)-OHhad been incomplete; the product was a circa 3:1 mixture of thesulfoxide Fmoc-Trp(2-NpsO)—OH and the sulfone Fmoc-Trp(2-NpsO₂)—OH.Proton NMR indicates a 1:1 mixture of the two compounds based on doublesets of signals for β-, α- and carboxyl-protons.

E) Preparation of Fmoc-Trp(2-Pmc)-OH

Method I: By Transferral of the Pmc-Group from Fmoc-Arg(Pmc)-OH

Fmoc-Arg(Pmc)-OH (0.5 g, 0.75 mmol) and Fmoc-Trp-OH (0.43 g, 0.1 mmol)was dissolved in 10 ml 100% TFA and heated at 30° C. for 1.5 hours.After evaporation of TFA, Fmoc-Arg-OH was removed by columnchromatography on silica gel with heptane/ethyl acetate 2:1 as mobilephase. Fmoc-Trp(2Pmc)-OH was isolated by preparative HPLC (C18, 70-100%B in 15 min., t_(R) 14.8 min, (A: H₂O+0.1% TFA; B: CH₃CN+0.1% TFA)).Isolated yield 130 mg (0.19 mmol, 25%).

¹N NMR (400 MHz, CDCl₃): δ 1.30 (s, 6H), 1.79 (t, 2H), 2.07 (s, 3H),2.43 (s, 3H), 2.48 (s, 3H), 2.59 (t, 2H), 3.03 (m, 1H), 3.25 (m, 1H),4.1-4.3 (m, 3H), 4.42 (m, 1H), 6.53 (d, 1H), 7.15-7.78 (m, 12H), 8.90(s, 1H).

Materials:

Fmoc-Arg(Pmc)-OH BACHEM no B-1670 Fmoc-Trp-OH BACHEM no B-1445/SENN no02019 Trifluoroacetic acid KEBO no 1.8341-100/Fluka no 91700

Method II: By Transferral of the Pmc-Group from phenylethylguanidyl-Pmc

2,2,5,7,8-Pentamethylchroman REFERENCES

-   Robert Ramage, Jeremy Green and Alexander J. Blake, Terahedron Vol.    47, No. 32, pp. 6353-6370, 1991.    Reaction:

Chemicals:

Substance Quantity MW mmoles eqv. Source 2,3,5-Trimethylphenol 50.03 g136.20 367.33 1.0 Fluka Isoprene 25.09 g 68.12 368.32 1.0 JannsenChimica ZnCl₂  5.94 g 136.29 43.58 0.12 Fluka Acetic acid   47 ml — — —KEBO labProcedure:

2,3,5-Trimethylphenol (50.03 g, 0.367 moles), isoprene (25.09 g, 0.368moles) and fused zinc chloride (5.94 g, 0.044 moles) was stirred withanhydrous acetic acid (47 ml) for 14 hours at room temperature. Thecloudy red coloured mixture was then gradually heated and it becameclear. Upon refluxing the reaction mixture turned black, and after 8hours of reflux it was cooled to room temperature. The reaction mixturewas poured into 250 ml water and the black oil separated. The water wasextracted with pentane (3×200 ml) and the combined organic phases washedwith Claisen's alkali (2×150 ml), water (3×250 ml) and brine (2×200 ml),dried over CaCl₂ and evaporated to a brown oil under reduced pressure.The crude product was distilled at 0.48 mbar affording the product as apale yellow liquid (36.90 g, 49% yield); b.p. 82-96° C. (0.48mbar); >95% pure (GC).

Results:

The product was isolated as a pale yellow liquid which solidified uponcooling in 49% yield.

¹H NMR (CDCl₃, 400 MHz): δ=1.30 (6H, s, 2×CH₃), 1.78 (2H, t, J=7 Hz,CH₂), 2.07 (3H, s, CH₃), 2.15 (3H, s, CH₃), 2.19 (3H, s, CH₃), 2.59 (2H,t, J=7 Hz, CH₂), 6.54 (1H, s, aromatic H).

¹³C NMR (CDCl₃, 400 MHz): δ=11.42 (CH₃), 18.91 (CH₃), 19.84 (CH₃), 20.49(CH₂), 26.97 (2×CH₃), 32.79 (CH₂), 73.10 (C(CH₃)₂), 116.67 (Ar—C),122.03 (Ar—C), 122.29 (Ar—C), 133.44 (Ar—C _(—), 134.70 (Ar—C), 151.72(Ar—C).

MS (GC/MS):

m/z=204(100), 189(14), 149 (91).

2.2.5.7.8-Pentamethylchroman-6-sulfonyl chloride

To a solution of 2,2,5,7,8-pentamethylchroman (3.39 g, 16.6 mmol) in 30ml dichloromethane at −8° C. was added under stirring chlorosulfonicacid (3.98 g, 34.2 mmol) in 30 ml dichloromethane within 3 minutes. Themixture was stirred at −8° C. for 15 minutes and at room temperature for2.5 hours. The reaction mixture was carefully shaken with 50 mldichloromethane and 100 ml ice a couple of times and the phasesseparated. The crude product contained circa 16% of starting material asjudged by ¹H NMR. When hot pentane was added to the crude product, adark oil was formed which was removed by decanting. The product was thenisolated by crystallisation from pentane as a light brown powder (2.80g, 9.3 mmol). Yield 56%.

¹H NMR (CDCl₃): δ 1.34 (s, 6H), 1.85 (t, J=7.0 Hz, 2H), 2.14 (s, 3H),2.60 (s, 3H), 2.62 (s, 3H), 2.68 (t, J=7.0 Hz, 2H).

2-Phenylethylguanidine hemisulfate

2-Phenylethylamine (8.49 g, 70.1 mmol) and S-methylisothiourea sulfate(9.43 g, 33.9 mmol) was dissolved in 100 ml destilled water. Air waspassed over the reaction mixture and through 50% NaOH (500 ml) and thenthrough 5% cuprous sulfate (250 ml). The reaction mixture was heated atreflux for 5 hours. Evaporation of the solvent yielded a white powder.The product was isolated by crystallisation from 96% ethanol, washedwith cold acetone and diethyl ether and dried in a desicator. Afterthree crystallisations, the product contained only minor amounts ofstarting material. The reaction yielded 61.5% (9.14 g)2-phenylethylguanidine hemisulfate.

¹H NMR (D₂O): δ 2.87 (t, J=6.6 Hz, 2H), 3.44 (t, J=6.6 z, 2H), 7.24-7.38(m, 5H)

Phenylethylguanidyl-Pmc:

-   Reaction: Ian Michael Eggleston, Ph.D. thesis, University of Oxford,    1990.    Reaction:

Chemicals:

Substance Quantity MW mmoles eqv. Source 2-Phenylethylguanidine 7.40 g212.24 34.87 1.5 B11-01 hemisulfate Pmc-SO₂—Cl 7.00 g 302.07 23.17 1.0B15-02 CH₂Cl₂  150 ml — — — KEBO labProcedure:

2-Phenylethylguanidine hemisulfate (3) (7.40 g, 34.87 mmoles) wassuspended in 6M NaOH (80 ml) and extracted into chloroform (2×80 ml).After evaporation of the solvent in vacuo the oily residue wasco-evaporated with benzene (2×10 ml). The free guanidine (3b) wasdissolved in 75 ml dichloromethane in a 250 ml round bottomed flaskequipped with a magnetic stirring bar and a 100 ml addition funnel withpressure equaliser. The funnel was charged with Pmc-SO₂—Cl (7.00 g,23.17 mmoles) dissolved in 75 ml dichloromethane. The equipment wasflushed with nitrogen, and the reaction was performed under a weaknitrogen flow. The round bottomed flask was cooled in an ice/water bath,and the Pmc-SO₂Cl solution added over a period of 20-25 minutes. Thereaction mixture was allowed to attain room temperature overnight. Thedichloromethane was evaporated in vacuo and the residue partitionedbetween water (100 ml) and ethyl acetate (120 ml). The organic layer wasthen washed with water (100 ml). Upon cooling of the ethyl acetate theproduct appeared as a white/pale yellow powder, which was filtered offand dried in vacuo.

Results:

3.32 g of a pale yellow powder was isolated. The yield of the reactionis 33%. Melting point: 145-147° C.

¹H NMR (CDCl₃): δ=1.30 (6H, s, 2×CH₃), 1.80 (2H, t J 7.0 Hz, CH₂), 2.09(3H, s, CH₃), 2.51 (3H, s, CH₃), 2.52 (3H, s, CH₃), 2.61 (2H, t J 6.6Hz, CH₂), 2.81 (2H, t J 7.0 Hz, CH₂), 3.43 (2H, m, CH₂), 6.21 (3H, broads, 3×NH), 7.12-7.25 (5H, m, aromatic protons).

MS: m/z=429(6), 204 (37), 149(100), 105(24), 92 (37).

Fmoc-Trp (2-Pmc)-OH:

Reaction:

Chemicals:

Substance Quantity MW mmoles eqv. Source Fmoc-Trp-OH 4.14 g 426.49 9.711.0 SENN N^(G)-(6-SO₂-Pmc)-2- 2.08 g 429.59 4.84 1.0 BEH-B17phenylethylguanidine Trifluoroacetic acid   25 ml — — — FlukaProcedure:

N^(G)-(6-SO₂-Pmc)-2-phenylethylguanidine (2.08 g, 4.84 mmoles) andFmoc-Trp-OH (4.14 g, 9.71 mmoles) was stirred with trifluoroacetic acid(25 ml) at room temperature for 2 hours. The reaction mixture was thenevaporated in vacuo and the residue partitioned between chloroform and 1M hydrochloric acid. By cooling of the chloroform solution the excess ofFmoc-Trp-OH could be removed by filtration.

The product was purified by flash chromatography (ethyl acetate/heptane,1:1).

Results:

The title compound was isolated as a white powder in 26% yield.

Materials:

Chlorosulfonic acid Fluka no 26388 Phenylethylamine Fluka no 77900S-Methylisothiourea sulfate Fluka no 67730 Fmoc-Trp-OH BACHEM no B-1445/SENN no 02019 Trifluoroacetic acid KEBO no 1.8341-100/ Fluka no 91700F) Preparation of Fmoc-2,5,7-tritertbutyltryptophan2,5,7-tritertbutyltryptophan

Tryptophan (4.00 g, 19.59 mmol), trifluoroacetic acid 98% (60 ml) andtert-butanol (15.54 g, 209.66 mmol) were mixed. The reaction mixture wasstirred at room temperature for 48 hours. The trifluoroacetic acid wasevaporated. The residue was suspended in 40 ml distilled water, and thepH adjusted to neutral with addition of sodium hydrogen carbonate. Thecrude product was obtained by filtration. Crystallisation from 50%ethanol afford the product as a white powder (85-90% pure).

¹H NMR (CDCl₃): δ=1.34 (9H, s, 3 CH₃), 1.46 (9H, s, 3 CH₃), 1.49 (9H, s,3CH₃), 7.45 (1H, s, CH arom), 7.18 (1H, s, CH arom), 5.29 (1H, s, NH).

Fmoc-2,5,7-tritertbutyltryptophan

The title compound was prepared as described for Fmoc-Trp (1-benzyl)-OH.

EXAMPLE 3 Bioactivity of Lactoferricin Analogs

Synthesis of the Analogs

All the peptides were synthesized on a 9050 Millipore Automatic PeptideSynthesizer using Fmoc protection and activation with pentafluorophenyl(Pfp)esters or in situ activation with the coupling reagent HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate). In the case of coupling with pentafluorophenylesters, 1-HOBt (1-hydroxy-benzotriazole) was added to catalyse thereaction, and when using the coupling reagent HATU the reaction was basecatalysed with DIPEA (diisopropylethylamine). All amino acids withreactive side chains were protected with acid labile protecting groupsand cleaved upon treatment with TFA (trifluoroacetic acid) containingscavengers. (See below for scavenger mixture). At the same time thepeptides were cleaved from the solid support on treatment with the TFAsolution.

A) Attachment of the First Amino Acid to the Solid Support whenSynthesizing all D-Peptides

The solid support PAC-PEG-PS (Peptide Acid-Poly Ethylene Glycol-PolyStyrene resin) (1 eq.) was mixed together with Fmoc-D-amino acid-OPfp (5eq.) and DMAP (dimethylaminopyridine) (1 eq.) in a small volume of DMF(dimethylformamide) and left to swell for 30 minutes. The solution wasthen stirred slowly for 4½ hours. Ac₂O (acetic acid anhydride) (2.5 eq.)and DMAP (0.1 eq.) were then added to the solution in order to acetylateany remaining hydroxyl groups on the solid support. The solution wasthen stirred for another hour. The solid support with the C-terminalamino acid attached was isolated by filtration and washed several timeson the filter with DMF. The solid support was then used in the synthesisof the target peptide on the 9050 Millipore Automatic PeptideSynthesizer.

B) Acetylation of the N-Terminal H₂N-Group Using Acetic Acid Anhydride

The peptide-resin complex was dissolved in a small volume of DMF andtreated with an excess of acetic acid anhydride (20 eq.) and DMAP (5eq.) for four hours while slowly stirring the solution with a smallmagnet. Complete acetylation was verified by a ninhydrin test/Kaiser'stest (see below).

C) Ninhydrin Test/Kaiser's Test

Less than 1 mg of the peptide-resin complex was treated with small equalvolumes of a 5% ninhydrin solution in ethanol, a solution of 80 g phenolin 20 ml ethanol and a solution of dried, distilled pyridine. Thereaction mixture was heated for two minutes at 110° C., and investigatedunder a microscope. (In this test a yellow reaction mixture indicatessuccessful acetylation, while a blue solution indicates still free aminogroups.)

D) Cleavage of Acid Labile Protecting Groups

Cleavage of acid labile protection groups and cleavage of the peptidesfrom the solid support was achieved using a mixture of 2% anisol, 2%ethandithiol (EDT), 2% water and 2% phenol in TFA, and with cleavagetimes of no more than four hours. The solid support was then removed byfiltration and the peptide precipitated in diethyl ether. The ethersolution containing TFA was removed using a pasteur pipette, and thepeptide was washed several times with diethylether and dried under highvacuum.

E) Purification

The peptides were purified by HPLC using a C18-reversed phase column (*)and a mixture of water and acetonitrile (both added 0.1% TFA) as mobilephase. Selected wavelength for detection of peptide fractions was 254nm.

(*) PrePak® Cartridge 25×100 mm. DeltaPak™ C18 15 μm 100 Å. (waterscorporation.)

F) Analysis

All peptides were analysed for impurities on an analytical HPLCC18-reversed phase column using a mixture of water and acetonitrile(both added 0.1% TFA) as mobile phase. The molecular weight of thepeptides were determined by positive ion electrospray ionization massspectrometry (VG Quattro Quadrupole).

Amino acid derivatives used in synthesis of both L- and D-analogs oflactoferricin Fmoc-AlaPEG-PS (solid support) Fmoc-Lys(tBoc)-OPfpFmoc-Arg(Pbf)-OH Fmoc-Met-OPfp Fmoc-Arg(Pmc)-OHFmoc-β-(2-naphthyl)-alanine-OH Fmoc-Asn(Trt)-OPfp Fmoc-Phe-OPfpFmoc-Cys(Acm)-OPfp Fmoc-Ser(tBu)-OPfp Fmoc-Gln-OPfp Fmoc-Thr(tBu)-OPfpFmoc-Glu(OtBu)-OPfp Fmoc-Trp-OPfp Fmoc-Gly-OPfpFmoc-Tyr(tBu)-OPfpFmoc-Leu-OPfp Amino acid derivatives were purchased from either Bachem,MilliGen/Biosearch (Division of Millipore) or PerSeptive Biosystems.Antimicrobial Activity of Alanine Scan Peptides Containing aTryptophan-Pmc Residue

During deprotection of acid labile protecting groups and cleavage of thepeptide from the resin with trifluoro acetic acid, a side reactioninvolving transfer of the Pmc (2,2,5,7,8-pentamethylchroman-6-sulphonylgroup) protecting group from arginine to the second position of theindole of tryptophan was observed. Isolation of these byproducts havebeen done, and the results from MIC analyses are given in Table 2. Thistable also shows the results of an alanine scan performed on LFB with noPmc groups.

During an alanine scan, a series of peptides are produced whereinsuccessive amino acids have been substituted by alanine.

The sequence of native bovine lactofericin from amino acids 17 to 31(LFB 17-31) isH₂N-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-COOH(SEQ ID No. 1).

TABLE 2 MIC results for alanine scan peptides with a Pmc group attachedto one of the two tryptophan residues. Results also shown for an alaninescan performed on LFB with no Pmc groups. Pmc Without Pmc MIC MIC MICMIC Peptide E. coli S. aureus E. coli S. aureus LFBA 1 8.75 10 70 >200LFBA 2 11.25 10 80 >200 LFBA 3 7.5  7.5 25 100 LFBA 4 15 27.5 70 >200LFBA 5 10 50(*) 120 >200 LFBA 6 25 17.5 >200 >200 LFBA 7 20  7.5 30 150LFBA 8 15 17.5 >200 >200 LFBA 9 10 12.5 55 >200 LFBA 10 20 22.5 140 >200LFBA 11 22.5 22.5 70 >200 LFBA 12 20 20 50 >200 LFBA 13 15 15 50 >200LFBA 14 15 17.5 25 160 LFBA 17-31 10 10 50 100 (*)MIC of 25 has beenobserved

The results show that a Pmc group attached to one of the tryptophanresidues increases the activity four times for E. coli. Even more markedis the effect on S. aureus. This gram positive bacteria was found to benearly totally resistant to all the alanine scan analogs, but shows nowa MIC between 10 and 22.5 μg/ml. This represents a ten fold increase inantibacterial activity relative to native LEB 17-31. The tryptophan-Pmcresidue, which is hydrophobic, seems therefore to increase the peptide;affinity for the hydrophobic parts of the bacterial cell membrane tosuch an extent that the antibacterial activity of the peptide is nolonger so sequence dependent as for the peptide without this residue.

Comparison of Antimicrobial Activity Between Native Bovine Lactoferricin(LFB 17-31) and Enantio-, Retro- and Retro-Enantio LFB 17-31 and TheseSame Peptides Incorporating a Tryptophan-Pmc Residue

Peptides containing a Pmc-group transferred from an arginine residue toa tryptophan residue was also isolated after synthesis of Enantio-,Retro- and Retro-Enatio LFB 17-31.

TABLE 3 MIC results for native bovine lactoferricin (LFB 17-31),Enantio-, Retro- and Retro-Enantio LFB 17-31 and for these peptides witha Pmc group attached to one of the two tryptophan residues. MIC %Hemolysis Peptide MIC E. coli S. aureus 10 μg/ml Native LFB 17-31 50 1002.6 Enantio LFB 17-31 7.5 60 3.05 Retro LFB 17-31 80 200 2.01Retro-Enantio LFB 17-31 6.25 80 3.31 LFB 17-31 Pmc 10 10 2.8 Enantio LFB17-31 Pmc 7.5(*) 100 3.17 Retro LFB 17-31 Pmc 10 10 2.5 Retro-EnantioLFB 17-31 7.5 12.5 5.28 Pmc *MIC of less than 5 has been observed.

The Enantio peptide, which is the exact mirror image of the nativepeptide, shows remarkable improvements in antibacterial activity. (Infact, this peptide shows the same activity as the native peptide LFB17-31 with a tryptophan-Pmc residue, in the case of E. coli.)Configurationally this means that an all-D-amino acid analog of LFB17-31 interacts better with the chiral phospholipids of the bacterialcell membrane than the native all-L-amino acid peptide LFB 17-31. It mayalso imply that this Enantio peptide is more resistant to degradableproteases of the bacteria.

The Retro peptide, with an inverted sequence in respect to LFB 17-31,shows no improvements in antibacterial activity, which is consistentwith the theory of the antibacterial activity of LFB 17-31 beingsequence specific. This peptide is not really an isomer of bovinelactoferricin since the amino acid sequence is totally different. Thelow antibacterial activity of this peptide does therefor not come as anysurprise.

A remarkably high antibacterial activity against E. coli was observedfor the Retro-Enantio peptide which, as already mentioned, adopts thesame α-helical conformation as the native peptide LFB 17-31, except thatthe amide bonds point in opposite directions. The all-L-amino acidstereoisomer, Retro LFB 17-31, shows low antibacterial activity. Thereason may be that all-D-amino acid peptides either interact morestrongly with the chiral phospholipids of the bacterial cell membrane orthat they are more resistant to proteases than their all-L-amino acidcounterparts.

The activity of the peptides against S. aureus is not as high asobserved for E. coli, indicating that the interactions of the peptideswith the lipopolysaccharide layer of gram negative bacteria might bestronger than the interactions with the lipid cell membrane of grampositive bacteria.

The activity of the tryptophan-Pmc containing peptides do not show thesame differences between all-D- and all-L-amino acid isomers as wasobserved for the peptides without the Pmc group. The effect of thetryptophan-Pmc residue seems to be more pronounced than theconfigurational effects found among the peptides without this residue,especially in the case of S. aureus. Most noticable is the tremendousincrease in activity of the Retro-Pmc peptide. The activity of thispeptide is increased eight times in the case of E. coli and more thanten times in the case of S. aureus just because of the tryptophan-Pmcresidue.

The improvements observed upon Pmc modification in the case of E. coliis negligible, but the modification increases the activity against S.aureus about six times. The gram positive bacteria are obviously morevulnerable towards tryptophan-Pmc containing peptides than their nontryptophan-Pmc containing counterparts.

Antimicrobial Activity of Tryptophan Modified Human (LFH), Porcine (LFP)and Caprine (LFG) Lactoferricin

The results from the alanine scan of bovine lactoferricin (LFB 17-31)showed that the two tryptophan residues in positions six and eight couldnot be substituted by alanine without a major loss of antibacterialactivity. Examination of the similar sequence parts of native LFH, LFPand LFG lactoferricin shows that these peptides lack the tryptophanresidue in position eight, but have during evolution conserved thetryptophan residue in position six. We have synthesized LFH, LFP and LFGanalogs with a tryptophan residue substituted in the position eight tosee if the antimicrobial activity of these peptides could be increased.The MIC values for the native sequences are given in Table 4, togetherwith the tryptophan modified peptides.

H₂N-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Trp-Asn-Met-Arg-Lys-Val-Arg-Gly-COOH

Sequence of modified human lactoferricin (LFHW8) Substituted tryptophanis high-lighted. (Arg-Trp)

H₂N-Ser-Lys-Cys-Tyr-Gln-Trp-Gln-Trp-Arg-Met-Arg-Lys-Leu-Gly-Ala-COOH

Sequence of modified caprine lactoferricin (LFGW8). Substitutedtryptophan is high-lighted. (Arg-Trp)

H₂N-Glu-Lys-Cys-Leu-Arg-Trp-Gln-Trp-Glu-Met-Arg-Lys-Val-Gly-Gly-COOH

TABLE 4 MIC results for tryptophan modified human (LFHW8) and caprine(LFGW8) lactoferricin. (MIC values for native LFB 17-31 and nativesequences of LFH and LFG are also listed for the sake of comparison.) %Hemolysis Peptide MIC E. coli MIC S. aureus 10 μg/ml LFHW8 110 >1000 2.5LFPW8 500 >1000 2.9 LFGW8 Y13 110 >1000 NT LFGW8 500 >1000 2.7 NativeLFB 17-31 50 100 2.6 Native LFH >1000 >1000 NT Native LFP >1000 >1000Native LFG 750 N.T 2.4 N.T = Not tested

Both LFHW8 and LFGW8 show improvements in activity against E. colicompared to the native sequences of the same peptides.

Antimicrobial Activity of LFH, LFP and LFG with a Tryptophan-Pmc Residue

During acidic cleavage of the peptide (either with or without the abovemodifications to the native sequence) from the resin and cleavage ofacid labile protecting groups, a byproduct with a Pmc-group attached toone of the tryptophan residues was isolated and analysed forantibacterial activity. The results are shown in Table 5.

TABLE 5 MIC results for LFH, LFG and LFP with a Pmc group attached toone of the two tryptophan residues, for LFC Pmc and LFH Pmc, the PMCgroup will be attached to the only available tryptophan. Peptide MIC E.coli MIC S. aureus LFG Pmc 25 25 LFH Pmc 25 50 LFHW8 Pmc 25 20 LFGW8 Pmc50 75 LFPW8 Pmc 20 50 LFHW8 Y13Pmc 25 20

As for all tryptophan-Pmc containing peptides analysed so far, thesepeptides generally show remarkable improvements in antibacterialactivity against both E. coli and S. aureus.

Antimicrobial Activity of Tryptophan Rich Analogs of BovineLactoferricin (LFB 17-31)

The alanine scan showed that the two tryptophan residues in the sequenceof bovine lactoferricin 17-31 were absolutely essential to theantibacterial activity of the peptide. Alanine substitution of any ofthese two residues led to a major loss of antibacterial activity. Thealanine scan also showed that the nonessential amino acids in thesequence of bovine lactoferricin 17-31 were the three residues Cys(3),Gln(7) and Gly(14). Based on this knowledge we therefore synthesized aseries of five tryptophan rich analogs of bovine lactoferricin 17-31with one, two or three of the nonessential amino acids substituted bytryptophan. This technique of performing an alanine scan and thenreplacing seeming non-essential amino acids with Tryptophan or otherbulky and/or lipophilic amino acids can be used to enhance thecytotoxicity of peptides generally, and is not limited to lactoferricin.The sequences of the tryptophan rich bovine lactoferricin analogs areshown below.

LFBW3:

H₂N-Phe-Lys-Trp-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-COOH

LFBW14:

H₂N-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Trp-Ala-COOH

LFBW3, 14:

H₂N-Phe-Lys-Trp-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Trp-Ala-COOH

LFBW3,7,14:

H₂N-Phe-Lys-Trp-Arg-Arg-Trp-Trp-Trp-Arg-Met-Lys-Lys-Leu-Trp-Ala-COOH

LFBW4,10:

H₂N-Phe-Lys-Cys-Trp-Arg-Trp-Gln-Trp-Arg-Trp-Lys-Lys-Leu-Gly-Ala-COOH

TABLE 6 MIC results for five tryptophan rich analogs of bovinelactoferricin (LFB 17-31) together with native LFB 17-31. Peptides MICE. coli MIC S. aureus LFB 17-31 50 100 LFBW3 20 20 LFBW14 20 25 LFBW3,14 10 10 LFBW3, 7, 14 20 20 LFBW4, 10 5 10

Substitution of nonessential amino acids in the sequence of LFB 17-31 bytryptophan residues improves the antibacterial activity of thesepeptides by at least two times that of the native sequence in the caseof E. coli and by four times in the case of S. aureus.

Peptide W3714, with three additional tryptophan residues (a total offive tryptophan residues in the peptide), has decreased activity. Thisis probably more a result of a solubility problem, this peptide beingless soluble in aqeuous solutions and therefore giving lowerconcentrations than calculated. This has been physically observed duringMIC testing procedures when the peptide tended to precipitate at highconcentrations.

EXAMPLE 4

Substituted magainin peptides have also been prepared. Native magainin 2has the following sequence:

MAG2

H₂N-Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Asn-Ser-COOH

Table 7 below shows the MIC results for the native peptide and a numberof modified peptides wherein single amino acid substitutions fortryptophan or phenylalanine at positions 16 or 19 have been made, with aresulting increase in antibacterial activity.

TABLE 7 Peptide MIC E. coli MIC S. aureus Mag2 20 >200 Mag2 W16 5-105-10 Mag2 W19 5-10 5-10 Mag2 F19 5-10 5-10

EXAMPLE 5 Antitumoral Effects of Different Peptides

Cyclic LFB 17-41 was from Morinaga, Milk Industri, Japan

Cytotoxicity

Different murine and human tumor cells (4×10⁶) were applied in 96-wellculture plates (Costar) in a volume of 0.1 ml RPMI 1640 medium. Peptidesolutions (0.1 ml) were added and the plates incubated at 37° C. for 30minutes, 4 hours or 24 hours. The cytotoxicity was measured using theMTT method (Mosmann et al., J. Immunol. (1986) 136, 2348-2357).

Electron Microscopy

Scanning Electron Microscopy (SEM)

For scanning electron microscopy, Meth A cells were cultivated in a 12well culture plate and treated with different peptides as describedabove. Cells were fixed in McDowell's fixative postfixated in 1% OSO₄,dehydrated and critical point dried according to standard procedures.The cells were examined in a Jeol JSM-5300 Scanning microscope.

Transmission Electron Microscopy (TEM)

Meth A cells were harvested from 12 culture plates by aspiration andfixed in McDowell's fixative overnight, followed by postfixation,dehydration and embedding in Epon Araldite according to standardprocedures. Ultrathin sections were cut on a Reichert Ultracut S andthen contrasted in 5% Uranyl acetate and Reynold's lead citrate.Sections were examined in a Jeol LEM-1010 transmission electronmicroscope.

Experimental Animals

Specific pathogen-free CB6F1 (Balb/c×C57 BL/6) female mice of about 8weeks of age were obtained from Charles River (Germany). The mice werekept on standard laboratory chow and water. Tumor bearing mice wereserologically screened for viral (LDH, CMV) and mycoplasmic infectionand in all cases tested negative.

Tumors

Meth A is a non-adhesive murine sarcoma cell line [Sveinbjørnsson et al,(1996) BBRC 223: 643-649] syngenic in Balb/c and was maintained in vitroin RPMI 1640 containing 2% Foetal calf serum. Cells in the growth phasewere harvested and washed in fresh medium and injected subcutaneouslyinto the abdominal region of the mice. Each mouse received a singleinoculation of 5×10⁶ viable tumor cells in RPMI 1640.

Results

In Vitro

Cytotoxicity

Lactoferricin B derivatives

A) Meth A

1. Cyclic and Linear LFB

The cytotoxic effect of cyclic and linear LFB (17-41) on Meth A cellswas studied. Linear LFB, with the cysteins protected with Acm, killedthe Meth A cells (1×10⁴/ml) effectively at concentrations higher than0.6 mg/ml after 4 h incubation (FIG. 2). Cyclic LFB, which is anenzymatically cleaved fragment of bovine lactoferrin effectively killedmore than 99% of the cells at concentrations higher than 0.8 mg/ml.

2. LFB Derivatives

LFB derivatives with different lengths and modifications were tested fortheir cytotoxic properties. Meth A cells were incubated with differentconcentrations of the different LFB derivatives, for ½ hour and 4 hours.As shown in FIG. 3, Unmodified LFB 17-31 had no significant cytotoxiceffect on the Meth A cells at concentrations up to 1 mg/ml after ½ hourincubation. In this experiment it had a weak effect at 1 mg/ml after 4hours incubation (FIG. 4). The PMC modified LFB 17-31 analog killed thetumor cells at concentrations higher than 500 μg/ml after ½ hourincubation. The same concentration was needed to achieve effectivekilling after 4 hours. Linear LFB (17-41) modified with Pmc was slightlymore effective than Pmc modified LFB 17-31.

In the figures, “−” denotes no Pmc modification and “+” denotes with Pmcmodification.

A shorter sequence LFB 20-29 modified with PMC killed more than 90% ofthe cells at 250 μg/ml. An LFB 17-31 analogue (alanine substitution inposition 8) that was modified with PMC and N-terminal Fmoc protected waseffective at concentrations higher than 100 μg/ml after ½ hour and at 50μg/ml after 4 hours. An Fmoc protected LFB peptide (Alanine substitutionin position 8) killed most of the cells at 250 μg/ml at ½ hour and 4hours (FIGS. 5 and 6). So it seems that a combination of Fmoc and Pmcmodification enhanced the cytotoxic effect of LFB more than each of thetwo modifications alone. The retro LFB analog was also tested. Theretro-Pmc-modified LFB 17-31 also possessed an enhanced cytotoxic effectcompared to unmodified LFB 17-31 (FIGS. 5 and 6).

B) Human Promelocytic Leukemia Cell Line HL60.

The cytotoxic effect of LFB 17-41 (PB), LFB 14-31 (P1), LFB 14-31 Pmc(P2), LFB (P3) 17-31 and LFB 17-31 Pmc (P4) on human HL 60 cells wasstudied. LFB 14-31 and LFB 17-31 showed no cytotoxic effect at theconcentration tested whereas LFB 17-41 possessed a weak concentrationdependant cytotoxic effect. The LFB 17-31 Pmc peptide induced a markedlystronger effect (appr. 5 fold higher) than the other peptides tested.See FIG. 7.

3. EM Studies

The SEM and TEM results show that the cell membranes are stronglydisrupted by lactoferricin peptides, resulting in effective release ofintracellular material. The lysis seems to be very rapid, i.e. withinminutes by the most effective peptides.

In Vivo

1. Tumor Regression

Murine Meth A Fibrosarcoma

After a single inoculation of 5×10⁷ viable Meth A cells, different LFpeptides were injected intratumorally (LFB-14-31, LFB 17-31 Pmc, 500 μgin a 50 μl dose; LFB 17-31, 1000 μg in a 50 μl dose), on day 7 and day10. LFB 14-31 was also injected intraperitoneally (PBI), 500 μg/ml.Saline only was injected in the control mice (50 μl) (K1, K2, K3). Thetumor diameter (mean of transversal and longitudinal) were measured withan electronic calipper.

The In Vivo Effect of LFB 17-31. LFB 17-31 Pmc and LFB on Murine MethAFibrosarcoma

As shown in FIG. 8, all three peptides tested, LFB 17-31 (PB), LFB 14-31pmc (P2), LFB 14-31 (P1), induced regression of the Meth A tumors, aftertreatment on day 7 and 10. “Diam.mm.” refers to diameter of the tumours.

Interestingly, tumors were also eradicated in the mice that were treatedintraperitoneally with LFB 14-31 (PBI). Mice treated with saline onlyare represented as K1, K2 and K3.

EXAMPLE 2 Murine melanoma B16F10

After a single inoculation of 5×10⁶ viable B16F10 murine melanoma cells,D-LFB A7 pmc-NH₂ was injected intra-tumorally in the tumors on day 10and 12 (500 μg/injection in 50 μl saline). Saline only was injected inthe control mice (50 μl). The tumor diameter (mean of transversal andlongitudinal) were measured every second day with an electroniccalipper.

The In Vivo Effect of D-LFB A7 Pmc-NH₂ on Murine Melanoma B16F10

As shown in FIG. 9, D-LFB A7 Pmc-NH₂ (pep) was able to effectivelyinduce regression of the solid tumors. The y axis represents thediameter of the tumour in mm. Three out of five were totally eradicatedafter only two injections. After six days after the first treatment, oneof the tumor started to grow again, and 10 days after the firsttreatment a second tumor started to grow.

2. Adaptive Immunity

After successful treatment of established MethA tumors, some mice werekept for one month before reinoculation of tumor cells as describedabove. In some of these mice a third inoculation of tumor cells wereperformed one month later than the second inoculation. No tumors wereestablished in these mice and the mice were kept for a longer periodwithout any effect on the normal condition of these mice.

EXAMPLE 6

The effect of chemical modification of a further moderately activepeptide has also been investigated. The stating peptide is a fragment ofbovine lactoferrin, which corresponds to residues 14-31 of the nativesequence (see Table 1 in FIG. 1 for the full sequence). Theantimicrobial activity in the form of MIC values against E. coli and S.aureus, the toxicity expressed as the concentration which caused 50%hemolysis (EC 50) and the anti-tumour activity in the form of the numberof μg/ml of peptide required to kill 50% of MethA cells for the peptidesare shown below in Table 8.

TABLE 8 MIC MethA MIC E. coli S. aureus EC 50 IC 50 Peptide μg/ml μg/ml(μM) μM/ml LFB 14-31 70 >250 >404 no activity LFB 14-31 PMC 15 20 24414.6 LFB 14-31 A2, 6, 10, 17 20 2.5 >440 165 LFB 14-31 A2, 6, 10, 17 PMC20 2.5 165 12.8 LFB 14-31 A2, 6, 10, 17R4 30 20 >438 75.8 LFB 14-31 A2,6, 10, 17R4 PMC 10 2.5 290 6.9 LFB 14-31 A2, 6, 10, 17R4, 11 >444 75.5LFB 14-31 A2, 6, 10, 17R4, 11 PMC 327 5.2 LFB 14-31 A2, 6, 10, 17F7R4 102.5 >440 30.2 LFB 14-31 A2, 6, 10, 17F7R4 PMC 10 2.5 20 7.7 LFB 14-31A2, 6, 10, 17F7K16L14 10 10 >440 28.1 LFB 14-31 A2, 6, 10, 17F7K16L14PMC 10 2.5 89 5.2

As before, the presence of the bulky/lipophilic group PMC on one or moreof the tryptophan residues enhances the antimicrobial and anti-tumouractivity. Interestingly, the presence of this artificial bulky andlipophilic group is able to selectively enhance bacteriocidal activity,activity against S. aureus generally being more enhanced than against E.coli.

EXAMPLE 7

Table 9 shows anti-bacterial activity and toxicity data for LFB basedpeptides incorporating a non-genetic bulky and lipophilic amino in placeof one of the amino acids in the native sequence. Further peptides alsoincorporate a group (PMC) which increases the bulk and lipophilicity ofone of the naturally occurring tryptophan residues.

TABLE 9 MIC MIC % % E. coli S. aureus Hemolysis Hemolysis Peptide μg/mlμg/ml 10 μg/ml 100 μg/ml LFB 50 100 2.6 3.47 LFB Bip3 10 10 LFB Bip6 2525 LFB Bip8 15 15 LFB Bip6, 8 10 5 LFB Bip3 PMC 37.5 2.5 LFB Bip8 PMC 255 LFB Tbt3 25 5 LFB Tbt3 PMC 37.5 10 LFB Tbt6 12.5 10 LFB Tbt6 PMC 37.510 LFB Tbt8 12.5 5 LFB Tbt8 PMC 25 5 LFB Tbt6, 8 25 5 LFB Nal6 20 75 2.24.4 LFB Nal6 PMC 25 20 2.8 17.8 LFB Nal6, 8 10 20 2.8 4.9 LFB Nal8 10 503 4.7 LFB Nal8 PMC 20 10 6.96 18.86 LFB NPS-O6 20 100 2.8 4.1 LFB NPS623 50 4.2 5.9 In the above table, Bip = biphenylalanine Tbt =tri-tert-butyltryptophan Nal = 2-naphtylalanine NPS =ortho-nitrophenylsulfinyl NPS-O = ortho-nitrophenylsulfonyl PMC =2,2,5,7,8-pentamethylchroman-6-sulphonyl

All peptides are LFB 17-31 and modifications thereof.

EXAMPLE 8

Experiments were performed to investigate the effect of PMC and varyingpeptide length on anti-tumour activity and toxicity (hemolyticactivity).

The results of these experiments are presented in Table 10 below.

TABLE 10 Meth A IC₅₀ (μM) RBC EC₅₀ (μM) Selectivity Peptide −PMC +PMC−PMC +PMC +PMC LFB 14-31 A_(2,6,10,17) 165 15 >440 118 8 LFB 14-30A_(2,6,10,17) >227 14 >454 184 13 LFB 14-29 A_(2,6,10) >235 18 >469 36720 LFB 14-28 A_(2,6,10) >248 12 >438 >438 >36

The presence of a PMC group on one or more of the tryptophan residues ofan LFB peptide significantly increased its anti-tumour activity and to alesser extent its hemolytic activity. Surprisingly it was found that byreducing the length of the peptide the selectivity, i.e. the anti-tumourverus the hemolytic activity of the peptide increased.

EXAMPLE 9

Modified peptides were prepared to investigate the effect of increasingthe number of tryptophan residues in margainin derived active peptides.The results of these modifications are shown in Table 11 below which hasMIC values for typical bacteria and shows anti-tumour activity by theμg/ml of peptide required to kill 50% of Meth A cells.

TABLE 11 Meth A MIC E. coli MIC S. aureus IC 50 Peptides (μg/ml) (μg/ml)(μg/ml) Mag 2 20 >100 100 Mag 2 W 19 7.5 10 9 Mag 2 W 6, 8 >100 >100 MSI24 5 5 >100 MSI 24 W7 5 5 26 MSI 24 W11 5 2.5 24 MSI 20 >100 MSI 20W6 * * 22 Tosmag 8 8 18 Tosmag W16 10 5 Tosmag W12, 16 5 10 Tosmag W6,12, 15, 17 15 10 23 Tosmag W5, 9, 13, 16, 20 >50 >50 23Mag 2:NH₂-Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Asn-Ser-COOH.Tosmag:NH₂-Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Arg-Lys-Phe-Gly-Arg-Ala-Phe-Val-Arg-Ile-Leu-Lys-Lys-Gly-COOH.MSI24:NH₂-Lys-Met-Ala-Ser-Lys-Ala-Gly-Lys-Ile-Ala-Gly-Lys-Ile-Ala-Lys-Val-Ala-Leu-Lys-Ala-Leu-NH_(2.)MSI20:NH₂-Lys-Val-Ala-Leu-Lys-Ala-Leu-Lys-Val-Ala-Leu-Lys-Ala-Leu-Lys-Val-Ala-Leu-Lys-Ala-Leu-NH_(2.)

For peptides having only moderate anti-tumour activity, the replacementof one residue with tryptophan significantly increases activity.

Tosmag is already highly cytotoxic and it is not surprising thatactivity is not significantly enhanced by substitution with tryptophan,as it is replacing one or more phenylalanine residues which arethemselves bulky and lipophilic.

Clearly, increasing the bulk and lipophilicity of the peptide too muchcan be counterproductive. This may be due to the fact that importantresidues are replaced or the fact that there is a limit on howbulky/lipophilic the side chains of the peptide should be.

EXAMPLE 10 Methods for the Preparation of Peptide Esters

Transesterification from Resin

Fully protected peptide esters can be obtained by base catalysedtransesterification from SASRIN™ and Merrifield-like resins. Good yieldshave been obtained with methanol and benzyl alcohol. The best resultswere obtained employing either KCN2, or LiBr/DBU as catalyst.

Standard Procedure for KCN-Catalysed Transesterification:

The peptide resin and the solvent employed have to be dried carefullybefore use, all have to withstand prolonged KCN-treatment.Transesterification will occur, even if the solubility of KCN is low;residual salt did not disturb. The peptide resin is suspended in amixture of the desired alcohol and the cosolvent, e.g.dimethylacetamide, (usually 1:1, 10 ml/g resin). After 30 min sufficientsolid KCN is added, so that a 0.08 M solution is obtained (or at leastsaturation). After stirring for 24 hours, the resin is filtered off andwashed with the cosolvent. The catalyst must be destroyed immediately,e.g. by rigourously shaking the filtrate with sufficient solid anhydrousFeCl₂. Iron blue will flock out, it is left to settle for approx. 30 minand filtered off. The filtrate may remain greenish. Further work-updepends on the solubility of the product, but it should be treated withwater: After removing alcohol and cosolvent, the residue is taken up inan organic solvent, e.g. ethyl acetate or chloroform, for furtheraqueous extraction to remove salts.

Direct Benzyl Esterification of N-Acylpeptides

(p-Hydroxyphenyl)benzylmethylsulfonium derivatives (HOBMX) easilygenerate benzyl cations, which converts N-terminal- and sidechain-protected peptides into their benzyl esters without racemization.

General procedure: The peptide and potassium carbonate are dissolved indichloromethane, and the mixture is stirred at room temperature. After10 min, HOBMCl is added to the solution and it is stirred for 8 hours.Inorganic salts in the reaction mixture are filtered off and thefiltrate evaporated in vacuo. The residue is dissolved in toluene andwashed with 0.5 M NaOH aqueous solution and then with water. The organiclayer is dried over anhydrous sodium sulfate and the filtrate evaporatedin vacuo.

EXAMPLE 11

A series of further modified peptides were prepared based on murinelactoferrin. In the following data (see Table 12), LFM refers toresidues 17-31 of murine lactoferrin. Shorter peptides are indicated bythe notation wherein e.g. LFM 17-24 represents an 8-mer peptidecorresponding to the amino acids at positions 17 through to 24 of murinelactoferrin.

The murine equivalent of LFB is generally much less active than itsbovine equivalent, however, by modifying the peptide in accordance withthe present invention peptides with greatly enhanced anti-bacterialactivity can be prepared. LFM does not possess a tryptophan residue atposition 8, unlike its more active bovine counterpart. The inventorshave identified this residue as important to the activity of LFB andthus this substitution of asparagine for tryptophan has been made. Thissubstitution alone did not significantly enhance the activity againstthe bacterial strains tested. Activity could be further enhanced bysubstituting one or both of the anionic residues at positions 1 and 9with unchanged alanine or more preferably a cationic residue such asarginine.

By incorporating further bulky/lipophilic residues, e.g. a tyrosineresidue at position 13 in place of the less bulky valine and/or bymodifying the tryptophan residue by incorporation of the more bulky PMCgroup, peptides with good antimicrobial activity could be made.

In addition it was surprisingly found that shorter peptides based onfragments of LFM when modified to introduce additional bulky/lipophilicamino acids e.g. tryptophan or tyrosine and to increase the overallcharge of the peptide by replacing native residues with cationicresidues such as arginine were particularly effective.

TABLE 12 MIC MIC % % E. coli S. aureus Hemolysis Hemolysis Peptide μg/mlμg/ml 10 μg/ml 100 μg/ml LFM >1000 >1000 2.3 3.1 LFM W8 >1000 1000 2.64.8 LFM W8 Y13 >1000 >1000 LFM A1 W8 750 >1000 2.4 3 LFM A1 W8 Y13500 >1000 LFM A9 W8 >1000 >1000 2.5 3.5 LFM A9 W8 Y13 >1000 >1000 LFMA1, 9 W8 200 >1000 2.8 3.7 LFM A1, 9 W8 Y13 150 >1000 LFM R1, W8 75 5002.8 3.48 LFM R1 W8 PMC >200 >200 LFM R1 W8 Y13 50 50 LFM R9 W8 500 >10003.1 4.59 LFM R9 W8 PMC 20 50 LFM R9 W8 Y13 150 1000 LFM R1, 9 W8 25 753.69 LFM R1, 9 W8 PMC 10 5 4.9 LFM R1, 9 W8 Y13 25 50 LFM A1 R9 W8 Y1350 200 LFM 17-24 R1, 2, 8 W3, 7Y4NH2 5 2.5 LFM 17-24 R1, 2, 8 W3, 7Y4NH2PMC 25 1-2.5 LFM 18-24 R1, 7 W2, 3, 6Y5NH2 10 0.5-1 LFM 17-25 A4R2, 8,9W3, 7Y1NH2 10 5 LFM 17-25 A4R2, 8, 9W3, 7Y1NH2 PMC 20 2.5 LFM 17-26A7R2, 8, 9W3, 4, 10Y1NH2 10 2.5

EXAMPLE 12

Table 13 below illustrates the effect of further chemical modificationswhich provide peptides in accordance with the invention.

TABLE 13 MIC % % MethA MIC E. coli S. aureus Hemolysis Hemolysis IC50Peptide μg/ml μg/ml 10 μg/ml 100 μg/ml μg/ml LFB 50 100 2.6 3.47 500 LFBPMC 10 10 2.8 4.4 120 LFB PMC6 10 10 3.4 6 148 LFB PMC8 18 10 3.6 9.79150 LFB 18-31 80 200 LFB 18-31 PMC 10 10 LFB 19-31 200 >250 LFB 19-31PMC 10 15 LFB 20-28 A4 >100 >100 0 1.68 500 LFB 20-28 A4 FMOC 120 LFB20-28 A4 FMOC PMC 35 LFB 20-28 A4 PMC 15 3.9 12.6 110 LFB 20-29 60 >1001.75 2.74 500 LFB 20-29 FMOC 5 10 10.3 28.2 140 LFB 20-29 FMOC PMC 22.560.2 50 LFB 20-29 PMC 10 10 5.6 18.9 160 LFB 20-30 40 >100 2.16 3.1 LFB20-30 PMC 15 10 5.54 15.8 LFB 20-31 100 200 LFB 20-31 PMC 10 10 LFB A170 >200 LFB A1 PMC 8.75 10 LFB A2 80 >200 LFB A2 PMC 11.25 10 LFB A3 25100 500 LFB A3 PMC 7.5 7.5 2 3.67 130 LFB A4 7.0 >200 LFB A4 PMC 15 27.5LFB A5 120 >200 LFB A5 PMC 10 50 LFB A6 >200 >200 2.78 3.27 LFB A6 PMC25 17.5 LFB A7 30 150 500 LFB A7 PMC 20 7.5 2.1 3.8 88 LFB A8 >200 >2002.8 3.45 500 LFB A8 FMOC 60 10 2.87 7.79 LFB A8 FMOC PMC 6.75 45.4 LFBA8 PMC 15 17.5 275 LFB A9 55 >200 LFB A9 PMC 10 12.5 LFB A10 140 >200LFB A10 PMC 20 22.5 LFB A11 70 >200 LFB A11 PMC 22.5 22.5 LFB A1250 >200 LFB A12 PMC 20 20 LFB A13 50 >200 LFB A13 PMC 15 15 LFB A14 25160 500 LFB A14 PMC 15 17.5 100 Unless otherwise indicated, LFBrepresents LFB 17-31.

EXAMPLE 13

Table 14 below illustrates the antibacterial activity as well astoxicity (% hemolysis) data for further peptides according to theinvention.

TABLE 14 MIC MIC % % Meth E. coli S. aureus Hemolysis Hemolysis A IC⁵⁰Peptide μg/ml μg/ml 10 μg/ml 100 μg/ml μg/ml LFB F4 20 200 2.4 3.2 LFBF4 PMC 20 20 LFB F4K1 20 200 LFB F4K1 PMC 10 10 LFB K1 60 100 LFB K1 PMC10 10 LFB W3 20 20 2.3 3.8 LFB W3 PMC >50 10 3.55 17.35 LFB W3, 14 10 103.1 5.1 LFB W3, 14 20 20 PMC LFB W3, 7, 14 20 20 4.02 66.1 LFB W3, 7, 1430 20 18.1 85.9 PMC LFB W4, 10 5 10 4.45 27.8 500 LFB W4, 10 20 20 2.2714.2 110 PMC LFB W14 20 25 3 4.1 LFB W14 PMC 25 10

EXAMPLE 14

Antibacterial peptides which are active against bacterial strains whichhave been shown to demonstrate resistance to other antibiotics arepotentially very useful peptides. Table 15 below gives the antibacterialactivity and toxicity data for some preferred peptides of the invention.MRSA is methicillin resistant S. aureus and MRSE is methicilin resistantS. epidermidis.

In Table 15, LFB=LFB 17-31 unless otherwise indicated. The previouslyidentified one and three letter codes are used and in addition, thefollowing N-terminal modifying groups are represented:

Bz=benzyl

CHx=cyclohexyl

Ad=adamantyl

TABLE 15 MIC MIC E. coli MIC S. aureus MIC MRSA MRSE EC 50 Peptide μg/mlμM μg/ml μM μg/ml μM μg/ml μM μg/ml μM Bz LFB Chx LFB >20 >9.2 2.5-51.1-2.3 Ad LFB  7.5-10 3.3-4.5 2.5-5 1.1-2.2 LFB PMC 6 10.0 4.3 2.5 1.12.5 1.1 >1000 >429 LFB A3 PMC 7.5 3.4 5.0 2.2 >1000 >449 LFB A7 PMC 20.08.8 15.5-20 6.8-8.8 ≧20 ≧8.8 17.5 7.7 >1000 LFB A3, 7 >20 >10.5 17.5-20 9.2-10.5 5.0 2.6 >1000 >525 LFB A3, 7 PMC 2.5 1.2 2.5 1.2 2.51.2 >1000 >461 LFB W3, 14 10.0 4.5 2.5 1.1 2.5 1.1 2.5 1.1 >1000 >453LFB retro PMC 10.0 4.3 5-7.5 2.1-3.2 >1000 >429 LFB enantio PMC 7.5 3.22.5-5 1.1-2.1 2.5-5 1.1-2.1 2.5 1.1 >1000 >429 LFB 20-30 PMC 15.0 8.310.0 5.5 LFB 17-27 A3, 7 R2, 11 W4, 10 Y1 NH2 10.0 5.9 2.5 1.5 0.5-10.3-0.6 2.5 1.5  700 ± 300  400 ± 200 LFB 17-27 A7 M3 R2, 11 W4, 10 Y110.0 5.7 0.5-1 0.3-0.6  510 ± 160 291 ± 91 NH2 LFB 17-27 A3, 7 R2, 11W4, 10 Y1 NH2    1-2.5 0.5-1.3 43 ± 8 22 ± 4 PMC LFB 17-27 A7 M3 R2, 11W4, 10 Y1  40 ± 10 20 ± 5 NH2 PMC LFB 14-31 A2, 6, 10, 17 20.08.8 >2.5 >1.1 5.0 2.2 >1000 >440 LFB 14-31 A2, 6, 10, 17 PMC 20.07.9 >2.5 >1.0 >1000 >394 LFB 14-31 A2, 6, 10, 17 R4 30.0 13.1 20.08.8 >1000 >438 LFB 14-31 A2, 6, 10, 17 R4 PMC 10.0 3.9 >2.5 >1.0 739 290LFB 14-31 A2, 6, 10, 17 R4, 11 12.5-15 5.6-6.7 2.5-5 1.1-2.2 >1000 >444LFB 14-31 A2, 6, 10, 17 R4, 11 PMC 2.5-5 1.0-2.0 2.5-5 1.0-2.0 823 327LFB 14-31 A2, 6, 10, 17 F7R4 10.0 4.4 >2.5 >1.1 >1000 >440 LFB 14-31 A2,6, 10, 17 K16L14 10.0 4.4 10.0 4.4 >1000 >440 LFB 14-31 A2, 6, 10, 17F7K16L14R4 110 LFB 14-31 A2, 6, 10, 17 F7L14Orn5, 4, 2.5 1.2 2.5 1.2 425202 8, 12, 15, 16 LFM R1, 9 W8 PMC 10.0 4.4 1.0 0.4 >1000 >435 LFM 17-24R1, 2, 8 W3, 7Y4H2 5.0 3.7 2.5-5 1.8-3.7 1.0 0.7 2.5 1.8 >1000 >733 LFM17-24 R1, 2, 8 W3, 7Y4NH2 PMC 20-50 34-53    1-2.5 0.6-1.5    1-2.50.6-1.5 2.5 1.5 120 ± 70  74 ± 43 LFM 18-24 R1, 7 W2, 3, 6Y5NH2 10.0 8.30.5-1 0.4-0.8   0.5-2.5 0.4-2.1 1.0 0.8 >1000 >829 LFM 17-25 A4R2, 8,9W3, 7Y1NH2 10.0 7.0 0.5-5 0.3-3.5 >1000 >697 LFM 17-25 A4R2, 8, 9W3,7Y1NH2 20.0 11.8 2.5 1.5 140 ± 90  82 ± 53 PMC LFM 17-26 A7R2, 8, 9W3,4, 10Y1NH2 10.0 6.2 1.0 0.6 2.5 1.5 880 ± 90 543 ± 56 LFM 17-26 A7R2, 8,9W3, 4, 10Y1NH2 200.0 106.0 2.5 1.3 2.5 1.3 150 ± 90  79 ± 48 PMC LFBBip 3 12.5 5.9 2.5-5 1.2-2.4 2.5 1.2 LFB Bip 6, 8 ≧12.5 ≧5.8 3.0 1.4 2.51.2 LFB Bip 3 PMC ≦25 ≦10.5 2.5 1.1 2.5-5 1.1-2.1 LFB Bip 8 PMC 15.0 6.32.5 1.1 2.5 1.1 LFB Nal 6, 8 10.0 4.8 20.0 9.6 5.0 2.4 >1000 >479 LFBTbt6 12.5 5.6 2.5 1.1    1-2.5 0.4-1.1 2.5 1.1 >1000 >448 LFB Tbt6 PMC37.5 15.0 2.5-5 1.0-2.0 5.0 2.0 410 ± 70 164 ± 28 LFB Tbt8 12.5 5.62.5-5 1.1-2.2   0.5-2.5 0.2-1.1 2.5 1.1 >1000 >448 LFB Tbt8 PMC 25-5010-23   2.5-7.5 1.0-3.0 5.0 2.0 290 ± 70 116 ± 28 LFB Tbt6, 8   25-37.512-18 2.5 1.2 5.0 2.3 2.5 1.2 230 ± 30 107 ± 14 LFB Tbt3 25.0 11.1 2.51.1 2.5 1.1 500 ± 40 223 ± 18

EXAMPLE 15

Table 16 below gives MIC values for a variety of peptides whichincorporate a proportion of D-amino acids.

MIC MIC MIC μg/ml μg/ml μg/ml Peptide Sequence* E. coli S. aureus MRSALFM 17-27 TYR-ARG-ALA- 10 2.5 A7R2, 8, 9W3, TRP-ARG-TRP- 4, 10Y1 NH2ALA-TRP-ARG- TRP-ARG-CONH2 tyr-ARG-ala-TRP- 7.5 7.5 2.5 arg-TRP-ala-TRP-arg-TRP-arg- CONH2 tyr-arg-ala-TRP- 7.5 5 2.5 ARG-TRP-ala- TRP-ARG-TRP-ARG-CONH2 LFM 18-24 ARG-TRP-TRP- 10 1 R1, 7W2, 3, ARG-TYR-TRP- 6Y5 NH2ARG-CONH2 arg-TRP-trp-ARG- 7.5 5 2.5 tyr-TRP-arg- CONH2 arg-trp-TRP-ARG-7.5 5 2.5 TYR-trp-arg- CONH2 *Upper case letters represents L-aminoacids, lower case letters denote D-amino acids

EXAMPLE 16 Cytotoxicity of the Peptides of the Invention

The cytotoxic effect of the peptides on different murine and human tumorcells were measured using the MTT method (Mosmann et al., J. Immunol.(1986) 136, 2348-2357). MTT is a soluble tetrazolium salt yielding ayellow solution when prepared in media or salt solutions lacking phenolred. Dissolved MTT is converted to an insoluble purple formazan bycleavage of the tetrazolium ring by dehydrogenase enzymes. This waterinsoluble formazan can be solubilized using isopropanol or othersolvents and the dissolved material is measured spectrophotometrically.The absorbance was measured as a function of concentration of converteddye.

The conversion of the soluble dye to the insoluble purple formazan isutilized in assays for measurement of cell proliferation. Activemitochondrial dehydrogenases of living cells cause this change, whiledead cells do not.

We used this assay to measure the degree of cell death caused bypeptides.

Cells:

Cells were maintained in RPMI-1641 medium containing 10% FBS, 1%L-glutamine and 0.1% penicillin and streptomycin. Cells to be used inthe assay were grown to confluency, trypsinated and split to single cellsuspension, counted and centrifuged at 1500 rpm for 10 min. The cellpellet was resuspended to a concentration of 4×105 cells/ml in RPMI-1640without FBS and L-glutamine (assay-medium). 100 ml of cell suspensionwas transferred to each well on a 96-well microtiter plate. The cellswere stimulated by adding 100 ml of various concentrations of peptidesdiluted with assay medium to each well. The final concentrations ofpeptide were for example: 5, 10, 20, 40, 60, 80, 100, and 200 mg/ml.Because there is a twofold dilution upon adding the peptide solution tothe wells containing the cell suspension, the peptide solution had to bemade twofold concentrated. As a negative control only medium was addedto the cells, and as a positive control (100% killing) 1% triton X-100was added. Following an incubation period of 4 h., 20 ml MTT dissolvedin PBS at a concentration of 5 mg/ml was added to each well, and theplate was incubated further for 2 h. 130 ml of the supernatant were thenremoved and 100 ml acid alcohol (0.04-0.1 N HCl in isopropanol) added toeach well to dissolve the dark blue crystals. The plate was placed on ashaker for 1 h and read spectrophotometrically at 590 nm in a microtiterplate reader using the Softmaxâ program.

Hemolytic Assay

The hemolytic activities of the peptides were determined using freshhuman red blood cells. 8 ml blood was taken from a healthy person. 4 mlblood was transferred to a polycarbonate tube containing heparin to afinal concentration of 10 U/ml, and the remaining 4 ml blood wastransferred to a glass tube containing EDTA with final concentration of15% EDTA. The erythrocytes were isolated from heparin-treated blood bycentrifugation in 1500 rpm for 10 min and washed three times withphosphate-buffered saline (PBS) to remove plasma and buffy coat. Thecell pellet was resuspended in PBS to make the final volume of 4 ml. Thepeptide was diluted to a concentration of 2 mg/ml and 0.1 mg/ml. Thepeptide was further diluted to the concentrations as stated in Table 1.For each tube PBS was added first, then RBCs and peptide solutions. Thehematocrit in the blood treated with EDTA was determined after 30 minwith Sysmex K-1000, and the resuspended RBCs were diluted into 10%hematocrit. RBCs in PBS (1%) with and without peptides (Table 18) wereincubated in a shaker at 37° for 1 hour and then centrifuged at 4000 rpmfor 5 min. The supernatant were carefully transferred to newpolycarbonate tubes and the absorbance of the supernatant was measuredat 540 nm. Baseline hemolysis was hemoglobin released in the presence ofPBS, and 100% hemolysis was hemoglobin released in the presence of 0.1%Triton X-100.

TABLE 17 Red Final peptide Peptide or blood Total concentration TritonX- cells PBS Volume Tube No. (μg/ml) 100 (μl) (μl) (μl) (μl) 1 Neg. 70630 700 (2 mg/ml control peptide) 2 Pos. 7 70 623 700 (2 mg/ml peptide)control 3 1000 250 50 200 500 (2 mg/ml peptide) 4 500 125 50 325 500 (2mg/ml peptide) 5 100 35 70 595 700 (2 mg/ml peptide) 6 50 17.5 70 612.5700 (2 mg/ml peptide) 7 10 70 70 560 700 (0.1 mg/ml peptide) 8 1 7 70623 700 (0.1 mg/ml peptide)

EXAMPLE 17 Solid Phase Peptide Synthesis

Initially the lactoferricin B used was a gift from Wayne Bellamy(Nutritional Science Laboratory, Morinaga Milk industry Co. Ltd, Japan).All the other peptides were synthesized on a 9050 Millipore AutomaticPeptide Synthesizer. Generally, in solid phase synthesis, peptide chainsare assembled from the carboxy terminus to the amino acid terminus. Thefirst (C-terminal) amino acid was covalently attached to an insolublesupport (the resin) by a linker (4-hydroxymethyl-phenoxyacetic acid).The remaining amino acids were added, one by one, until the peptidesequence was completed.

Using the Fmoc method, the α-amino end of the amino acid was temporaryprotected by the base labile 9-fluorenylmethoxycarbonyl (Fmoc) group.Not only the α-amino group of the amino acid was protected. Some of theamino acids have reactive side chains which are necessary to protectduring the synthesis to prevent side reactions. These protecting groups,except for cysteine, were acid labile and cleaved upon treatment withTFA (trifluoroacetic acid) and scavengers (see below).

Prior to the synthesis, to the solid support PEG-PS (Poly EthyleneGlycol-Poly Styrene resin) was added a small volume of DMF(dimethylformamide) and left to swell for 30 minutes. Packed into acolumn, the Fmoc group was removed by treatment with a 20% piperidinesolution in DMF. The incoming protected amino acid was now able to bindto the free amino end of the resin-linked amino acid with its carboxyend. However, coupling or acylation does not occur spontaneously, thecarboxylate must be activated. This was achieved by the use ofpreactivated amino acids, such as pentafluorophenyl (Pfp) esters, oramino acids with free carboxylates able to react with the couplingreagent HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluoro-phosphate) in situ. Using Pfp esters, 1.3 eq. of HOBt(1-hydroxy-benzotriazole) was added to catalyse the reaction, while whenusing the coupling reagent HATU, the reaction was base catalysed with2.4 eq. of DIPEA (diisopropylethylamine). A four-fold excess ofactivated amino acids was generally employed. The amino acids weredissolved in the activator solution in sufficient quantity, ascalculated by the Express-Peptide program.

Amino acids were then delivered to the support-bound amino acid/peptidewith fully deprotected α-amine group, and recycled through the loopmixed to achieve peptide bond formation. The capacity of the resins usedscaled from 0.15 to 0.23 mmol/g, meaning available binding sites for theincoming amino acids, wherefrom the amount of activator equivalents wascalculated. The standard coupling cycle for amino acids was 30 minutes,with the exception of arginine, isoleucine, threonine, tyrosine, valine,and the amino acids coupled thereafter, requiring 60 minutes. Extendedcoupling times for these amino acids were chosen because of their largeside chains which are known to cause sterical hindrance during thecoupling reaction. Once coupling was complete, the excess amino acidsolution and reaction by-products were removed by washing with DMF. Thenext cycle begun with deblocking of the α-amino group of the N-terminalamino acid. The process of α-amino group deblocking followed by couplingwas repeated for as many cycles as necessary to assemble the desiredpeptide.

After the synthesis was complete, the column material was transferred toa funnel and washed with methanol (3×) and dichloromethane (2×) Thecleavage of the acid labile side chain protecting groups and cleavage ofthe peptides from the solid support was achieved using a mixture of 2%anisol, 2% ethandithiol, 2% water and 2% phenol in TFA, and withcleavage times of no more than four hours. The solid support was thenremoved by filtration, the filtrate concentrated under a high vacuum andthe peptide precipitated in diethyl ether. The ether solution containingTFA was removed using a pasteur pipette, and the peptide was washedseveral times with diethyl ether and dried under a high vacuum.

Amino Acid Derivatives:

Fmoc-L-Ala-OPfp

Fmoc-L-Arg(Pmc)-OPfp

Fmoc-L-Cys(Acm)-OPfp

Fmoc-L-Gln-OPfp

Fmoc-L-Glu(OtBu)-OPfp

Fmoc-L-Gly-OPfp

Fmoc-L-Ile-OPfp

Fmoc-L-Leu-OPfp

Fmoc-L-Lys(tBoc)-OPfp

Fmoc-L-Met-OPfp

Fmoc-L-Phe-OPfp

Fmoc-L-Ser(tBu)-OPfp

Fmoc-L-Trp-OPfp

Fmoc-L-Tyr(tBu)-OPfp

Fmoc-L-Val-OPfp

Amino acid derivatives: Amino acid derivatives were purchased fromeither Bachem, MilliGen/Biosearch (Division of Millipore) or PerSeptiveBiosystems. Phenol was purchased from Fluka, and anisole was purchasedfrom Sigma. DMF, PIP, DIPEA TFA and PEG-PS resin were all purchased fromPerSeptive Biosystems.

EXAMPLE 18

Table 18 below shows the anti-tumour activity and toxic data for a LFB14-31 derivative incorporating either of two non-genetic bulky andlipohilic amino acids in place of a Trp or incorporating a group (Pmc)which increases the bulk and lipophilicity of one of the naturallyoccurring Trp or Phe residues.

TABLE 18 RBC EC₅₀ Meth A EC₅₀ Variable Peptide (μM) (μM) LFB14-31A2, 6,10, 17 >440 165 LFB14-31A2, 6, 10, 17Bip4 336 23.4 LFB14-31A2, 6, 10,17Pmc 165 12.8 LFB14-31A2, 6, 10, 17Tbt9 25.6 9.5

The presence of either of the three non-genetically modifications on aLFB 14-31 derivative significantly increased its anti-tumor activity.The Tbt modified peptide however possessed the highest hemolyticactivity among the three modified analogs tested.

EXAMPLE 19

Table 17 below shows the anti-bacterial and anti-tumour activity andtoxicity of further peptides according to the invention. In particular,the substitutions show how replacement of tryptophan may result inpeptides with advantageously low toxicity (activity against red bloodcells and normal fibroblasts).

TABLE 19 Meth A IC₅₀ Mic E-coli Mic S. Aureus RBC EC₅₀ FibroblastSubstituion Peptide (μM)(4 h) (μM) (μM) (μM) IC₅₀ (μM) LFB14-31A_(2,6,10,17)F₇K₁₆L₁₄R₄ 6.6 2/4 2 110 17 Alanine W3-A3 LFB14-31A_(2,3,6,10,17)F₇K₁₆L₁₄R₄ 24.1 15 10 >463 190 W9-A9 LFB14-31A_(2,6,9,10,17)F₇K₁₆L₁₄R₄ 16.2 10 5 382 46.3 W11-A11 LFB14-31A_(2,6,10,11,17)F₇K₁₆L₁₄R₄ 11.1 10 >2.5 278 46.3 W9,11-A9,11 LFB14-31A_(2,6,9,10,11,17)F₇K₁₆L₁₄R₄ 110.1 30 30 >489 >489 Lysine W3-K3 LFB14-31A_(2,6,10,17)F₇K_(3,16)L₁₄R₄ 230 >451 230 W9-K9 LFB14-31A_(2,6,10,17)F₇K_(9,16)L₁₄R₄ 13.5 30 10 >451 58.7 W11-K11 LFB14-31A_(2,6,10,17)F₇K_(11,16)L₁₄R₄ 7.9  5 <2.5 >451 30.7 W9,11-K9,11 LFB14-31A_(2,6,10,17)F₇K_(9,11,16)L₁₄R₄ >300 >463 >463 Isoleucine W3-I3 LFB14-31A_(2,6,10,17)F₇I₃K₁₆L₁₄R₄ 9 2/4 2/4 323 20 W9-I9 LFB14-31A_(2,6,10,17)F₇I₉K₁₆L₁₄R₄ 12  5 <1 26 W11-I11 LFB14-31A_(2,6,10,17)F₇I₁₁K₁₆L₁₄R₄ 6 2/5 <1 15 W9,11-I9,11 LFB14-31A_(2,6,10,17)F₇I_(9,11)K₁₆L₁₄R₄ 22 26 W3,9-I3,9 LFB14-31A_(2,6,10,17)F₇I3,₉K₁₆L₁₄R₄ 36  5 5 >470 108 W3,11-I3,11 LFB14-31A_(2,6,10,17)F₇I_(3,11)K₁₆L₁₄R₄ 16   2.5 5 413 45 W3,9,11-I3,9,11LFB 14-31A_(2,6,10,17)F₇I_(3,9,11)K₁₆L₁₄R₄ 47   2.5 10 >487 280 F7-A7LFB 14-31A_(2,6,10,17)F₇K₁₆L₁₄R₄ 34.6 15 10 >455 288.9

1. A method of treating tumours in a patient in need of such treatmentcomprising the administration to said patient of a peptide selected fromthe group consisting of a cytotoxic 7-25 mer peptide with three or morecationic residues, modified at its N-terminal amino acid by a lipophiliccyclic group comprising 5 non-hydrogen atoms; a cytotoxic 7-25 merpeptide with three or more cationic residues, modified at its C-terminalamino acid by a lipophilic group comprising at least 6 non-hydrogen; andesters, amides, salts and cyclic derivatives of said peptides.
 2. Themethod of claim 1 wherein said peptide has 7-15 amino acids.
 3. Themethod of claim 1 wherein said lipophilic cyclic group comprises 6non-hydrogen atoms.
 4. The method of claim 1 wherein said lipophiliccyclic group is a polycyclic group wherein the cyclic groups are fusedor bridged.
 5. The method of claim 1 wherein said peptide has a di- ortri-alkylated N-terminal amine.
 6. The method of claim 1 wherein saidlipophilic cyclic group is an alkyl group having the formulaCH₃(CH₂)_(n), wherein n is between 5 and
 20. 7. The method of claim 1wherein said lipophilic cyclic group is an alkyl group having theformula CH₃(CH₂)_(n), wherein n is between 10 and
 12. 8. The method ofclaim 1 wherein said lipophilic cyclic group is an acyl group havingbetween 6 and 21 carbon atoms.
 9. The method of claim 1 wherein saidlipophilic cyclic group is an acyl group having between 11 and 13 carbonatoms.
 10. The method of claim 1 wherein said lipophilic cyclic group(R) is attached to the N-terminal amine via a linking moiety selectedfrom a carbonyl group (RCO), carbamate (ROCO), a linker which forms urea(RNHCO or R₂NCO) or by a linker which forms a sulfonamide, boronamide orphosphoramide.
 11. The method of claim 1 wherein said peptide has alipophilic group comprising at least 4 non-hydrogen atoms attached toits C-terminal carboxy group.
 12. The method of claim 1 wherein saidlipophilic group has 10 or more non hydrogen atoms.
 13. The method ofclaim 1 wherein said lipophilic group incorporates a cyclic group. 14.The method of claim 1 wherein said lipophilic group (R) is attached tothe C-terminal carboxy group via a linking moiety selected from OR,NH—R, NR₂ and B—(OR)₂.